Reactions of Nucleic Acids and NucleoDroteins with Formaldehyde1

Reactions of Nucleic Acids and Nucleoproteins with Forma Idehyde'

I

M. YA. FELDMAN Institute of Molecular Biology, Acrtdemy of Scie)ices, iMoscoto, V.S.S.R.

I. Introduction . . . . . . . . . . . . . 11. Interaction of Formaldehyde with Bases, Nuclcosides and Nucleotides . . . . . . . . . . . . . . A. Primary Reactions : Formation of Methylol Derivatives, R--CH?OH . . . . . . . . . . . . . 13. Secondary Rciictions: Formntion of Metliylcne Dinucleotides, R-CHL-R' . . . . . . . . . . . . 111. Interaction of Formaldchydc with Polynuclrotidcs . . . . A. Synthetic Polynucleotidcs . . . . . . . . . B. Ribonucleic Acids . . . . . . . . . . C. Deoxyribonucleic Acids . . . . . . . . . . D. Effect of Secondary Structure . . . . . . . . E. Effect of Formaldehyde on thc Functional Activity of Nuclcic Acids . . . . . . . . . . . . . . IV. Interaction of Formaldcliyde with Nucleoproteins . . . . . A. Formation of Mcthylene Bridges in thc Reaction of Protein with . . . . . . . Formaldeh ydc B. Effect of Formaldehyde on Nuclcoprotcins . . . . . V. Use of Reactions of Nucleic Acids and Nucleoproteins with Formaldehydt . . . . . . . . . . . . A. Structural and Functional Studics of Nidcic Acids . . . B. Inactivation of Viruses by Formoldchydc in Vaccine Production. C. Effect of Formaldehyde on the Genetic Aplmratus of thc Cell . VI. Related Reactions and Thcir Effects (:I* Compsred to Formaldc. . . . . hyde Reactions) . . . A. Miscellanc~ousAldehydes . . . . . . . . . . B. Difunctional Alkylating Agents . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . .

.

.

. .

. . . .

.

. . . . .

1 3 3 10 15 15 16 19

m

28 30 30 33 35 35 36

38 40 40 42 44 44

1. Introduction Three main reiLsons for the chemical modification of nucleic acids can be singled out in a review of the information available on modifying agents. These agents are used for inactivations of various kinds (such as inactivation of viral RNA or of cytostatic action), for directed functional Translntcd by A. L. Pumpinnsky, Moscow. 1

2

M. YA. FELDMAN

changes of nucleic acids in vivo (mutagenesis, oncogenesis) and for the elucidation, through resulting modification, of structural and functional characteristics peculiar to synthetic or native polynucleotides. Some of these agents can be useful for one, two or all three of these purposes. One of the few agents that serves all three of them is formaldehyde. It is widely used as an inactivator of viruses to obtain vaccines ( I ) and is reported to exert a cytostatic (carcinostatic) effect (a). It is also one of the most promising mutagenic agents affecting multicellular organisms (3, 4 ) . Formaldehyde is used extensively in structural and functional studies of nucleic acids as an agent not so much for causing denaturation as for preventing renaturation, and as a fixator of nucleic acids and nucleoproteins in electron microscope and sedimentation investigations. The use of nucleic acid reactions with formaldehyde has outstripped our knowledge of their mode of action. In many cases a chemical mechanism was postulated for some biological or physical effect that could not plausibly be substantiated, and such diverse products as Schiff bases (5-7), monomethylol derivatives (RNH-CH,OH) (8,9), (R-N=CH,) mono- and dimethylol (R-N(CH,OH) *) derivatives ( l o ) , as well as methylol and methylene (RNH-CH,-NHR) compounds (11-13) were suggested., Recently some progress in the structural study of formaldehyde interaction products with nucleotides and nucleic acids has been made. Evidence has been presented that the reactions proceed according to the following scheme:

A '

0 -NH RNH, RNH-CHZOH

A '

+ CHZO F? 0 -N-CHzOH + CHzO + RNH-CHIOH + RNHZ + RNH-CHz-NHR

4-HzO

(14 (1b) (2)

Reaction ( l a ) proceeds with the participation of the -CO-NH- grouping of pyrimidine and purine heterocycles. In reaction ( l b ) , formaldehyde interacts with the exocyclic amino groups of AMP, GMP and CMP. Reaction (2) involves aminopurines only. This review is the first attempt to sum up the data available on the interaction of formaldehyde with nucleic acids and nucleoproteins with particular emphasis on the evidence for the formation of various structures and the molecular mechanisms of biological and other effects of formaldehyde. *Useful information on formaldehyde chemistry is provided by Walker in his book (14).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

3

II. Interaction of Formaldehyde with Bases, Nucleosides and Nucleotides

A. Primary Reactions: Formation of Methylol Derivatives, R-CH OH The reactions described in this section were discovered by FraenkelConrat in 1954 ( 5 ) . They result in labile noncrystallizable compounds found only in solutions also containing starting compounds (formaldehyde and the nucleic base or its derivative). The reaction product is readily dissociated on simple dilution. However, it was later shown (15, 16) that these reactions are not the only ones to occur when formaldehyde acts on bases or nucleotides. When the reaction mixtures are allowed to stand for many days, the labile (hydroxymethyl or methylol) derivative reacts with the starting base present in the mixture [Eq. ( 2 ) ] to form a methylene bis-compound as the end product. The formation of methylene derivatives does not affect the investigation of primary (methylol) derivatives, the former appearing much later. Some methylene derivatives are precipitated quantitatively ( 1 5 ) . 1. FUNCTIONAL GROUPS

The question, what functional groups react with formaldehyde, was formerly settled by comparing different bases, nucleosides and nucleotides. The comparison was made by means of two tests involving the spectral changes of bases under the action of formaldehyde ( 5 ) and the quantitative estimation of the formaldehyde added (12). Reactions were essentially carried out in neutral buffered aqueous medium a t room temperature for 24-48 hours. I n this time, the primary reaction was almost completed whereas the products of the secondary reaction were still practically absent. The resulting data are presented in Table I. I n all cases studied, compounds containing amino groups reacted with formaldehyde with a marked change in the ultraviolet spectrum, the maximum shifting to longer wavelengths by 3-5 nm and its intensity rising by about 20%. Similar spectral changes occur when formaldehyde acts on deoxyribonucleotides containing amino groups (6). The data presented in the last column of Table I indicate the participation in the reaction not only of the exocyclic groups, but also of the NH-groups in position 9 (or 7) of purines and, possibly, in position 1 of pyrimidines (e.g., in hypoxanthine, 1,3-dimethylxanthine, 2,6,8-trichloropurine, uracil). No reaction takes place if the hydrogen atoms in these positions are replaced by methyl or ribosyl residues (inosine, 1,9-di- and 1,3,9-trimethylxanthine, uridine) .

4

M. YA. FELDMAN

TABLE IR INTERACTION OF PYRIMIDINE AND PURINE DERIVATIVES WITH FORMALDEHYDE IN NEUTRAL AQUEOUS SOLUTION AT ROOM TEMPERATURE AND RELATIVELY Low CONCENTRATION OF CHsO. PRIMARY REACTION (24-48 HOURS) Spectral changes in the presence of 1-2% CHzO (6) Increase of

Compound

emax

(%I

Formaldehyde bound (mole per Shift of XmRx to 100 moles of longer wave- purine or pyrimilength dine derivative)b (nm1 (18)

Pyrimidine derivatives

Uracil Thymine 1,3-Dirnethyluracil Uridine Uridylic acid Cytosine 2-Amino-4-ox yp yrimidine (isocytosine) 2-Aminop y rimidine Cytidylic acid

None None

-

None None -

1 3 None None

-

-

16

3

4 2 0 0

11

5 10

-

Purine derivatives

Adenine Adenosine Adenylic acid Guanylic acid Hypoxanthme 1,3-Dimethylxanthine 2,6,8-Trichloropurine Inosine 1,g-Dimethylxanthine 1,3,9-Trirnethylxanthine

23 19 22

5 -

5 5 5 5

-

10 9 13 10 9 24 0

-

1 0

-

Nucleic acida

RNA (TMV) DNA (thymus)

29 None

20

3 None

-

Reproduced by permission of Elsevier Publishing Co., Amsterdam. concentrations of reagents were 0.01 M. The free formaldehyde was determined (17,18) and the bonded aldehyde was calculated as the difference between the overall and free formaldehyde. 4

* The initial

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

5

It was later shown that the experiments listed in Table I failed to take into account the reaction of formaldehyde with ring NH-groups in position 3 of pyrimidines and 1 of purines, seemingly owing to the relatively low formaldehyde concentration used. At high CH,O concentrations, the absorption spectra of nucleosides with no NH,-group change slightly, thus indicating that they react with formaldehyde. The ultraviolet maxima for uridine and thymidine in 3.3M formaldehyde solution (pH 6.9) decrease by 2% and are shifted to longer wavelength by 2 nm (18). The spectra of uridylic and polyuridylic acids (18, 19) as well as that of inosine (20) reveal similar changes. These changes are believed to be caused by addition of formaldehyde to the N-3 of uridine and the N-1 of inosine. The respective N-1 and N-9 positions must be excluded as they are blocked by ribose residues. That the spectral changes cannot result from formaldehyde addition to C-5 was shown by experiments with thymidine (18), whose spectral changes with formaldehyde were similar to those for uridine, in spite of its blocked C-5 position. The reported spectral data (18-20) do not allow for possible formaldehyde interaction with hydroxyl groups of bases and ribose. The spectral analysis of pseudouridine with different formaldehyde concentrations suggested (18) that, in compounds containing two unsubstituted NH-groups in the ring (pseudouridine, uracil, thymine), both groups react. The participation of ‘
PH

FIG.1 . Titration of 0.00227 M adenine hydrochloride solution, with (B) and without (A) added formaldehyde (0.34%) at 19”C,by aqueous sodium hydroxide (21). By permission of the Chemical Society, London.

6

M. YA. FELDMAN

(9.8) is shifted upward ( 2 1 , 2 2 ) .I n the first case, the pH depression arises from the interaction of the free amino groups with formaldehyde, similar to the reaction of formaldehyde with other amines, in particular with amino acids. I n the region of the acidic dissociation constant (pKg), arising from the g-NH-group, the pH is raised by the following mechanism:

\NH

/

+F

\NHI:

/

[see (19-23)]. The pK values (ca. 4) of cytidylic ( 2 4 ) , adenylic and polyadenylic (25) acids are also lowered by formaldehyde, additional proof of the interaction of formaldehyde with the amino groups of nucleotides. A marked shift of the titration plot to higher p H is observed in the pI(, region of the NH-groups of inosine (20) and of uridylic and polyuridylic acids (19). This fact, together with spectral evidence, proved the interaction of formaldehyde and compounds containing the HN3-C40

I I

grouping in the pyrimidines or the HN1-CEOgrouping in the purines.

I I

A reaction of formaldehyde with the hydroxyl groups of uracil in the tautomeric form is excluded because the pH of solutions of compounds containing “acidic” hydroxyl groups, such as phenol, is not affected on formaldehyde addition whereas such a change is typical of compounds containing amino and imino groups ( 2 1 ) .Titration data do not indicate a participation of the N-1 of adenine in the formaldehyde reaction (22, 25), nor does spectral evidence (see Section 11, A, 2). Formaldehyde addition to the C-5 of uracil (26) (resulting in the formation of a crystalline product, 5-hydroxymethyluracil) takes place in the presence of 0.42 M KOH (73 hours a t 50°C) or 0.5 M HCl (reflux for 25 hours). To prepare 5-hydroxymethyl derivatives of uridylic and cytidylic acids, even more rigorous conditions were used (27). The results on the reaction of formaldehyde with sugars in neutral aqueous medium (14) do not unequivocally exclude the interaction of formaldehyde with the hydroxyl groups of ribose and deoxyribose to form labile semiacetals (R-0-CH,OH) . There is, however, no evidence that such an interaction does actually take place. In any case, low concentrations of CH,O (0.01 M ) do not react in a measurable amount with D-ribose and D-2-deoxyribose (12). Under ordinary conditions, if the nucleotides are not destroyed, their phosphate groups are unlikely to

7

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

react with formaldehyde [on formaldehyde reactions with phosphoric acid, see (14)1. It can thus be concluded that a t a pH close to neutral, formaldehyde reacts with the NH,- and NH-groups marked in formulas I-IV with asterisks.

Rib

Rib

(n)

(1)

NH;

0

I

Rib

(III)

Rib

(IV)

This scheme can be considered as proved for uridine ( I ) , cytidine (11) and adenosine (111) but not as conclusively for guanosine (IV). When the ribose residue is replaced by hydrogen, the glycosyl nitrogen a t position 1 of the pyrimidine bases and position 9 of the purine bases can also be attacked by CH,O. 2. SUBSTANTIATION OF THE FORMATION OF MONOMETHYLOL

DERIVATIVES

The identification of the primary products of the reaction of formaldehyde with bases and nucleotides is made difficult by their lability. There exists only indirect, spectral and kinetic, proof that these products are monomethylol derivatives (>NCH,OH) . a. Spectral Evidence. Michelson and Grunberg-illanago (65) synthesized N6-hydroxyethyladenylic and poly ( No-hydroxyethyladenylic) acids and found their spectra to be similar to those of adenylic and polyadenylic acids treated with formaldehyde (1% CH,O, pH 6.8, 2.5 hours a t 37"). The similarity shows that the formaldehyde reaction with adenylic and polyadenylic acids most likely gives rise to hydroxymethyl (monomethylol, -NH-CH,OH) derivatives rather than to Schiff bases (-N=CH,) , As No-hydroxyethyladenylic and poly (Ns-hydroxyethyl-

8

M. YA. FELDMAN

adenylic) acids do not react with formaldehyde (the spectra remaining unaffected on addition of CH,O), formaldehyde is unlikely to react with adenylic and polyadenylic acids to give N6-dialkyl derivatives. Similar results were reported by Scheit (28). 2',3'-O-Isopropylidene cytidine reacted with formaldehyde (pH 7, 37°C) to give a product that could be separated chromatographically from the starting product on a thin silica gel layer. The ultraviolet spectrum of this product was found to be similar to that of isopropylidene N6-hydroxyethylcytidine. The latter compound failed to react with formaldehyde. It follows from this that formaldehyde reaction with isopropylidene cytidine leads to isopropylidene N6-hydroxymethylcytidine (V) rather than to a bis (N6hydroxymethyl) derivative. HN-C&OH

I

H,C0 x 0 CH,

The formaldehyde addition products with AMP (25) and isopropylidene adenosine (28) have spectra similar to those of No-methyladenosine (maximum shift to long wavelengths and increased absorption in the maximum as compared to adenosine in both acidic and alkali media), but different from those of N1-methyladenosine. This fact leads again to the conclusion that in the formaldehyde reaction alkylation proceeds a t the 6-amino group rather than N-1. b. Kinetic Evidence. The results from the kinetic study of the primary reaction only (at the early stage of the interaction) are in fair accord with the conception that one NH,-group reacts with one formaldehyde molecule (6, 8, 29). The same result is also always observed from the analysis of pH shifts and spectral changes due to formaldehyde interaction with ring NH-groups (18-23).In the latter case, the l-to-1 ratio points unambiguously to the formation of the structure >NCH,OH, because another derivative allowing for the same ratio, the Schiff base, cannot be produced in the reaction with NH-groups. The proof for the formation of monomethylol derivatives obtained

9

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

(SO) by the kinetic study of the overall conversion of adenosine in the primary and secondary reactions is presented in Section 11, B, 1.

3. PROPERTIES OF METHYLOL DERIVATIVES

It has been noted above that these compounds are extremely labile and dissociate into the starting components on simple dilution of the reaction mixtures. Since this dissociation takes place under chromatographic conditions, few workers (31, 28) have succeeded in observing the separation of hydroxymethyl derivatives from the starting compounds on chromatography or electrophoresis. When equimolar concentrations of reagents are used, the amount of formaldehyde bound to nucleotides and bases is much lower than would be expected from considerations of equivalency (see Table I). Spectral changes (8, 22, 19, 18) and the shift of titration plots (24,22,19) increase up to a certain limit as the formaldehyde concentration is raised. This dependence on concentration is used to estimate the equilibrium constants presented in Table 11. It will be seen that various authors have obtained almost the same results by different methods. The reaction constants for the ring NH-groups are much lower than those for the exocyclic amino TABLE I1 EQUILIBRIUM CONSTANTS (K = [RCH20H]/[RH].[CH20])OF PRIMARY FORMALDEHYDE REACTIONS WITH NUCLEOSIDES AND NUCLEOTIDES Compound reacting with CHzO

Reaction conditions

Adenosine

pH 4.8; room temp.

Adenosine

pH 7.3; room temp.

Adenylic acid 5'-AMP 5'-dAMP 5'-CMP 5'-dCMP Uridine 5'-UMP

pH 7.3; room temp.

Inosinic acid 4

pH 7.05; room temp. pH 7.05; room temp. pH 7.05; room temp. pH 7.05; room temp. pH 6.6; 23°C pH 4.7; 20°C 20°C 40°C pH 6.6; 23°C

Method Quantitative analysis of the reaction products Quantitative analysis of the reaction products Quantitative analysis of the reaction products UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry UV spectrophotometry Titration Titration UV spectrophotometry

(1.mole-1)

K

Reference

15.8

30

12.3

a

10.9 11.4 11.3 16.6 15.5 2.5

2.6 2.42 1.35 1.7

K is calculated from data (18) listed in the last column of Table I.

(I

8 8 8 8 18 19

19 19

18

10

M. YA. FELDMAN

groups. No results for uracil (23) and guanylic acid (8) are given, for further studies have indicated that formaldehyde may add to each of these substances in two positions rather than one, as previously suggested (see Section 11, A, 1). In the reactions with the NH-groups of nucleosides and poly(U), the equilibrium is reached in less than 30 seconds (18).At room temperature and in a neutral medium, the primary reactions of CH,O with NH,-groups are close to equilibrium in 1-2 days, The adenosine reaction with formaldehyde a t pH 4.8 and 20°C attains equilibrium in 72 hours (SO). Kinetic analysis of spectrophotometric data reveals (69) that as the temperature is raised from 30" to 45°C the formaldehyde interaction with adenosine and poly(A) in a neutral medium is accelerated in both the direct and reverse reactions, the reaction rates increasing from 5 to 10 times. The values of the equilibrium constants for the primary formaldehyde reaction with bases and nucleotides fall with rising temperature as first observed in the titration of the NH,- and 9-NH-groups of adenine (22, 23) and the NH-group of uridylic acid (19).The decreased K for 5'-UMP when the temperature is raised from 20" to 40°C is exemplified in Table 11. This evidence was substantiated spectrophotometrically for mixtures of formaldehyde with AMP and CMP over a wide temperature range, from 15" to 85"C, a t p H 7.5 (32) as well as for CH,O and adenosine (30"-45") (29). Higher temperature never affects the extent of the reaction markedly, and in experiments with G M P (32) and poly (A) (29), the reaction is practically unaffected. There are insufficient results on the influence of hydrogen ion concentration to warrant any general conclusions. Lowering the pH to 2.4 to the acidic side or raising it to 10.8 to the alkaline side does not increase formaldehyde bonding to adenine, adenosine, adenylic acid, hypoxanthine, or thymine. On the contrary, even a t these pH values the amount of formaldehyde bound is lower than that in neutral solution (12). From spectrophotometric results (8), the primary CH,O reactions with nucleotides involving the NH,-group, equilibrium is attained a t the same rate over the pH range of 4 to 8, whereas at pH 10 it is sharply increased. It was shown spectrophotometrically that changing the concentration of the neutral phosphate buffer does not affect ( I I ) , or only weakly affects (8),the interaction of formaldehyde with nucleotides.

B. Secondary Reactions: Formation of Methylene Dinucleotides, R-CH,-R' Numerous stable products of the reaction of formaldehyde with purine and pyrimidine compounds have been isolated and identified, such as

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

11

5-hydroxymethyl derivatives of uracil (26), uridylic and cytidylic acids (27) (the reactions proceeding in the presence of alkali or acid with heating) , 7-hydroxymethyluric acid (condensation in the presence of KOH) (33), methylene bis (2-aminopyrimidine) ( 3 4 ) , methylene bis (6arninouracil) (12, 55) (synthesized in neutral medium with or without heating) (see reviews (36-38)) . The patent literature describes the preparation of resins through the action of formaldehyde on di-, tri- and tetraaminopyrimidines under different conditions (39-41). However, if the choice of bases is confined to those characteristic of nucleic acids and the reaction conditions are limited to those compatible with biological experiments, only those purine compounds will be of interest that react with formaldehyde to give stable condensation products, methylene derivThese compounds are dealt with in this atives of the type R-CH,-R’. section. Methylene bis-compounds are formed in the reactions of formaldehyde with adenine, adenosine and adenylic and guanylic acids (15,16). A reaction mixture containing AMP, GMP and formaldehyde gives, together with methylene bis-adenylic and methylene bis-guanylic acids, methylene adenine-guanine dinucleotide ( 1 6 ) . Pyrimidine components of nucleic acids fail to form stable condensation products with formaldehyde under similar conditions (16). 1. METHYLENE BIS-COMPOUNDS OF THE ADENINE SERIES

a. Methylene bis-Adenine ( V I ; R = H ) and Methylene bis-Adenosine ( V I ; R = Ribose Residue), These compounds are formed when adenine or adenosine and formaldehyde are kept a t room temperature and pH 4.5 for many days ( 1 5 ) . This pH is optimal. The product yield is diminished in a more acidic medium as well as in neutral and weakly alkaline media.

I

I

R

R

(VI)

The proof of the formation of methylene bis-compounds is as follows. Two moles of adenine or adenosine react with one mole of formaldehyde ( 1 5 ) . The dependence of the yield of reaction product (during time t ) upon the logarithm of the initial concentration of formaldehyde is graphically represented by a bell-shaped curve (Fig. 2) (SO). Both criteria are specific for methylene bis-compounds.

12

M. YA. FELDMAN

3.0 2.5 +

E

b 2.0 n 0

+

L

1.5

L11

I

1 " 1.0 V I

rn

0.5 0

-3

-2

-1

0

Logarithm of initial molar CH,O concentration

FIQ. 2. Influence of CH,O concentration on the formation of methylene bisadenosine (SO).pH 4.8, 20°C. Curve 1: initial adenosine concentration 0.01 M, incubation for 85 days; Curve 2: 0.015 M adenosine, 53 days.The experimentally found points are placed on the curves derived from theory, taking K N 16 l.mole-', kz 1.4 l.mole-'.day-', K being the equilibrium constant of reaction (3) and kl the rate constant of reaction (4). Reproduced by permission of Nauka, USSR.

The curves presented in Fig. 2 need special explanation. The experimental data are in good accord with theoretical plots corresponding to the reaction sequence RH R-CHIOH

+ CH20 @ R-CHIOH + R H R-CHZ-R + HzO +

(3) (4)

Raising the formaldehyde concentration gives an increased amount of methylol derivative (R--CHIOH) and, conversely, a decreased amount of adenosine (RH) in the reaction mixture. Ideally, the maximal rate of the secondary reaction is when [R-CH20H] = [RH] . With an initial adenosine concentration of 0.01-0.015 M, conversion of 50% of adenosine to its methylol derivative necessitates a formaldehyde 7 (the equilibrium constant for the primary concentration of ~ 0 . 0 M reaction being c 16). With this initial concentration of formaldehyde (0.07M), the secondary reaction runs a t the maximal rate (Fig. 2), whereas higher or lower formaldehyde concentrations lead to slower rates of methylene bis-adenosine formation.

13

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

Two important reaction characteristics should be borne in mind when constructing the ideal curve and in plotting the experimental results, 1. The primary reaction practically reaches equilibrium before the secondary reaction starts (prior to the appearance of a measurable amount of methylene bis-derivative) . With an initial adenosine concentration of 0.01-0.015 M and a formaldehyde concentration of 0.014.1 M, methylene bis-adenosine is reported (30) to appear (as a negligible precipitate) only after the solutions have been kept at room temperature (pH 4.8) for 6-8 days. This is in contrast to the primary reaction (Section 11, A, 3 ) , which reaches equilibrium in about 70 hours. On the other hand, if the methylene bis-derivative is readily formed, the reaction fails to give a bell-shaped curve for concentration dependence, as exemplified by the interaction of CH,O with 6-aminouracil (30). 2. No essential shift in equilibrium (3) is observed during reaction (4),which requires an equimolar amount of RH and R-CH,OH on the left and right sides of Eq. ( 3 ) , respectively. The superimposition of the theoretical and experimental plots (Fig. 2) is considered (30) as definitely proving the reaction to lead to methylene bis-adenosine, the monomethylol derivative being an intermediate product. Other possible reactions (see Section I) cannot give a symmetrical bell-shaped curve of concentration dependence. The ultraviolet absorption spectra of methylene bis-adenine and methylene bis-adenosine are characterized by the shift of the maximum to longer wavelengths and an increased absorption a t the maximum as compared with adenine and adenosine, respectively (Table 111). These spectral changes are typical of 6-N-alkyl substitution, being stronger for TABLE I11 ULTRAVIOLET MAXIMA OF METHYLENE BI&OMPOUNDS Compound Methylene bis-adenine Methylene bis-adenosine Methylene bis(adenosine 2’(3’)-phosphate) Methylene bis(guanosine 2’(3’)-phosphate)b

Reference

Xmax

fmnxa

Solvent

(nm)

(X10’)

0 . 0 5 M HCl 0 . 1 M NaOH 0.25 M KOH Acetate, pH 5

28 1 277 and 284 272 272

17.2 14.8 and 13.4 16.8 -

16 48

Acetate, pH 5

255

13.0

43

16 43

Absorbance at the maximum, calculated for 1 M monomer (or 0.5 M methylene bis-compound). Under the same conditions, Xmax = 252 nm and emnr = 12100 for guanosine-2‘(3’)phosphate. (1

14

M. YA. FELDMAN

methylene than 6-N-hydroxymethyl derivatives (see Section 11, A, 1 ) . The nuclear magnetic resonance spectrum of methylene bis-adenine (42) (100 Mc/sec, 10% solution of the compound in 1M NaOD) reveals three signals (singlets) due to protons bonded to carbons. Two signals common to adenine (44, 45) with a chemical shift of 8.46 and 8.27 ppm 6 are due to H-2 and H-8 (2 and 8 positions remaining thus unsubstituted). The third signal (5.51 ppm) has the same intensity as the first two (corrcsponding to two protons) and is to be accounted for by the inethylene group binding two adenine residues. The chemical shift of 5.51 ppm conforms to the position of the methylene group between two exocyclic nitrogens, HN-CH,-NH. (The methylene group bonded to two nitro-

I

I

gens involved in the heterocycles is expected to cause a chemical shift of about 7 ppm.) Methylene bis-adenine and methylene bis-adenosine share several common characteristics (15), such as insolubility in water and organic solvents, decomposition in mineral acids with liberation of formaldehyde and base, low chromatographic mobility and high melting points. b. Methylene bis-Adenylic Acid (VZ; R, Phosphoribosyl Residue). This acid may be isolated chromatographically from the mixture of AMP and CH,O ( 1 6 ) . The reaction goes to completion in 15 days (at room temperature and pH 4.8) and gives rise to a very low yield of C H , ( A P ) ~ not exceeding 6 4 % of theory. When chromatographed according to Cohn (46) on Dowex 1 in a formate system, methylene bis(adenosine 2'(3')phosphate) is separated from 2'- and 3'-AMP to give three isomers (16) with different positions of phosphate groups ( P-2'-Ado-CH2-Ado-2'-P, P-2'-Ado-CH2-Ado-3'-P, P-3'-Ado-CHz-Ado-3'-P) . Dephosphorylation of the three products results in methylene bis-adenosine. When chromatographed on DEAE-cellulose in a concentration gradient of NaCl (pH 5 ) , CH2(Ap) separates without dissociating into isomers (4s). 2. METHYLENE BIS-COMPOUNDS OF THE GUANINE SERIES These products have been less extensively studied than those of the adenine series. The low solubility of guanine and guanosine hinders the investigation of their interaction with formaldehyde. Dissolution of guanosine a t 70" (0.01 M, pH 4.8) and immediate addition of formaldehyde (0.1 M ) leads to a stable (at 36°C) solution that gives rise, in 10-15 days, to a slowly growing precipitate ( 4 7 ) .This precipitate has not yet been identified but its properties resemble those of the methylene bisderivatives of adenine and adenosine. It is insoluble in water, on paper chromatography in alcohol-acid mixtures it follows guanosine with an R, close to that of methylene bis-adenosine, and on hydrolysis in 1 M

15

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

HC1 a t 100" it gives off CH,O. It might thus be suggested that the precipitated product is methylenc bis-guanosine. The reaction product of GMP and CH,O, methylene bis-guanylic acid, is formed as slowly as is CH2(Ap)a(16).The effect of formaldehyde concentration is expressed by a symmetrical bell-shaped curve. On paper chromatography, CH, (Gp) , runs far behind GMP. Ion-exchange chromatography on Dowex 1 makes it possible to separate methylene bis(guanosine 2'(3') -phosphate) into isomers that are eluted over the same range of formic acid concentrations (approximately from 2.6 M to 4.0M ) as are C H , ( A P ) ~isomers, though the CH,(Ap), and CH,(Gp), isomers can be separated quite sharply.

3. MISCELLANEOUS DIMERS A reaction mixture containing CH,O and two nucleotides (AMP and GMP) yields a product not found in mixtures involving CH,O and only one of the two nucleotides. Thus the reaction product should have the structure P-Guo-CHa-Ado-P, which agrees with base composition (A and G in a 1-to-1 ratio) as well as by the bell-shaped curve for the concentration dependence. Other mixtures, such as UMP CMP + C H 2 0 and AMP G M P UMP CH,O have also been studied, but failed to show any additional condensation products (16, 48).

+

+

+

+

111. Interaction of Formaldehyde with Polynucleotides

A. Synthetic Polynucleotides The only results available on the interaction of biosynthetic polynucleotides with formaldehyde are concerned only with the labile primary products formed in the early stage of the reaction. On treatment of polynucleotides with [W]formaldehyde (0.1%) in neutral solutions a t room temperature for 16-20 hours followed by precipitation of polynucleotides with ethanol (11), the label is bound appreciably only in those polynucleotides containing amino groups: poly(A) and poly ( C ) , but not poly(U) and poly(1). When poly(A) (49, 29) and poly(C) (6, 50) are treated with formaldehyde, their spectra change just as do those of corresponding monomers. Poly (N6-hydroxyethyladenylic acid) does not react with formaldehyde ( 2 5 ) , nor does N6-hydroxyethyladenylic acid. The similarity in spectra indicates that the action of CH,O on polymers gives rise to the same products obtained on CH,O treatment of the corresponding monomers, that is, aminomethylol derivatives R-NH CH,OH. This

.

16

M. YA. FELDMAN

accords the decreased pk' of poly(A) in formaldehyde solutions, to 3.0 (25j.

The reactions of formaldehyde with poly(U) and UMP have many common characteristics (18, 19), such as identical spectral changes in solutions with a high formaldehyde concentration, increased pK, ready completion of the reactions that rcach equilibrium in less than 30 seconds, and similar equilibrium constants whose values are similarly lowered a t higher temperature (evidence for UMP is given in Section 11,A, 1).It is evident that formaldehyde is bound to the uracil residues in poly(U) just as it is in free uridylic acid. The resulting N3-hydroxymethyl derivatives are extremely labile, and it apparently suffices for their quantitative dissociation to remove the free formaldehyde by repeated polynucleotide precipitation. This, as well as the low initial formaldehyde concentration, seems to explain the almost complete absence of bound [14C]formaldehyde in the experiments (11) with poly(U) and poly(1).

B. Ribonucleic Acids As far back as 1901 the patent literature carried reports on the treatment of nucleic acid with formaldehyde (51). Yet it was only after Fraenkel-Conrat indicated a change in the ultraviolet absorption spectrum of TMV-RNA in the presence of formaldehyde ( 5 ) (see Table I) that biologists and biochemists directed their attention to the reaction of RNA with formaldehyde. In the same work (5)-that is, two years before it was discovered that TMV-RNA is infectious (56, 53)-he suggested that the formaldehyde inactivation of viruses is due to the action of formaldehyde on the nucleic component rather than on the protein. In 1958, Staehelin (11) suggested that the TMV-RNA reaction with formaldehyde gives rise both to labile products that dissociate on dialysis and to stable derivatives. He also suggested that by analogy with the formaldehyde interaction with proteins, this reaction can be considered as resulting in labile aminomethylol compounds and stable methylene compounds R-CH,-R'. However, Staehelin's results could be interpreted quite differently and did not necessarily point to the formation of two types of compounds (9). Yet, his suggestion about the successive formation of methylol and methylene derivatives was substantiated both by low molecular models (see Section 11, B) and by direct study of the reaction of RNA with formaldehyde (6.4,16). 1. METHYLOL DERIVATIVES

The main methods used to study the primary reaction products of RNA and formaldehyde are spectral analysis and quantitative estimation of bound [ I T ] formaldehyde. However, neither of them provides any

17

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

information about CH,O addition to cyclic nitrogen (N-3 of uracil and N-1 of guanine residues). Minute changes in the ultraviolet spectrum caused by this addition are obscured by the large spectral changes due to the reaction of exocyclic amino groups with formaldehyde. Quantitative cstiniation of the bound label requires the removal of the free [14C]forinaldehyde, which seems to be accompanied by the complete dissociation of extremely labile products of the addition of formaldehyde to NHgroups in the heterocycles. Therefore, it is only possible to discuss formaldehyde interaction with exocyclic amino groups of ribonucleic acids. The formation of aminomethylol derivatives (R-NHCH,OH) may be judged by characteristic spectral changes (see Table I ) .These spectral changes are almost completely reversible on prolonged (4 days) dialysis ( 5 5 ) . The dynamics of the formation of methylol derivatives in the formaldehyde reaction with amino groups of RNA a t room temperature and pH 4.6 is shown in Fig. 3 (curve 1 ) . To simplify the understanding of primary reactions, the equilibrium shift due to the very slow formation of methylene derivatives is not considered. The same time is required for equilibrium to be reached (about 100 hours) a t pH 7.6 (9) whereas the aminomethylol derivative of adenosine is formed in 72 hours a t p H 4.8 and 20°C (SO).

!

8

y I

100

-

200

300

3

I

4 I

400

I

I

500

600

Hours

FIG.3. Reaction of tRNA with "CH,O with time. Measurements were made by different workers (9, 56) under similar conditions. The nonfractionated yeast tRNA reacts with "CH20 in a weak acidic solution (pH 4.3-4.7) a t low ionic strength a t room temperature. Curve 1: Total number of nucleotides labeled in 0.1 M "CH,O without Mg" (tRNA concentration on nucleotides, 0.003 M) ; Curve 2: the same in the presence of Mg2+( 9 ) ; Curve 3: tRNA nucleotides participating in the formation of methylene cross-links L0.2 M "CH,O, 0.02 M (as nucleotides) tRNA1; Curve 4 : same as 3 in the presence of Mg2+( 6 6 ) . By permission of John Wiley & Sons, Inc., Nrw York, and Elsevier Publishing Co., Amsterdam.

18

M. YA. FELDMAN

Equilibrium is attained much more quickly in the primary RNA reaction with formaldehyde as the temperature is raised. Thus, a t 63" in 1 M formaldehyde (0.1 M phosphate, pH 8.5), the reaction with amino groups is nearly complete (about 85%) in 10 minutes (32). I n a 0.5% solution of CH,O (in 0.001 M phosphate buffer) the reaction is over in 10 hours a t 80°C (57). Such an acceleration of the reaction a t higher temperatures cannot be considered as due solely to the destruction of the secondary structure of RNA. In Section 11, A, 3 evidence was presented (29) on the increased rate of both the direct and reversed reactions when CHrO mixtures with adenosine or poly(A) are heated in neutral medium. Judging by the equilibrium constants, the NH,-groups of RNA react with formaldehyde just as do the amino groups of free nucleotides. According to Boedtker ( 3 2 ) , under denaturation conditions (at 63°C) the NH,-groups of RNA are responsible for the bonding of an amount of l'CH,O that corresponds to K N 11.0, i.e., to a value close to those of equilibrium constants for the formaldehyde reactions with the NH,groups of free nucleotides (see Section 11, A, 3 ) . Changes in pH over the range of 5-8 do not affect the amount of 14CH,0 bound to RNA (11). At pH 4.6, [14C]formaldehyde is bound to the amino groups of tRNA in a somewhat smaller amount than a t pH 7.6 (9), with 40% of the tRNA amino groups reacting with formaldehyde in the former case (see Fig. 3) and 48% in the latter. The difference is accounted for by a partial dissociation at pH 4.6 of the amino groups of cytidylic acid (pK,, = 4.5)'. 2. METHYLENE DERIVATIVES Methylene bis-adenylic acid, methylene bis-guanylic acid and methylene adenine-guanine dinucleotide have been isolated chromatographically (on Dowex 1, formate) from alkali hydrolyzates of formaldehyde-treated rRNA (rabbit liver) (16). Every methylene bis-nucleotide is in turn divided into isomers differing in the positions of phosphate groups whose exact location in the isomers is, however, as yet uncertain. No stable condensation products of formaldehyde with pyrimidine residues of RNA have been discovered (16). The dynamics of the formation of methylene his-derivatives in the tRNA reaction with formaldehyde is shown in Fig. 3. The same kinetic plots as in Fig. 3 are also obtained on '*CH,O action on the rRNA of rabbit liver and TMV-RNA (16, 56). In our experiments (16, 43) a t pH 3.5, from 27 to 30% of purine nucleotides of the rRNA are involved in the formation of methylene bridges; a t pH 4.8, the percentage is 12-17; a t p H 7 to 8.5, only 6-7 (15-24 days, room temperature, 0.2 M CH,O; ionic strength of buffer, 0.1). The reaction becomes faster as the formaldehyde

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

19

concentration is raised to 0.2 M (pH 4.8, room temperature, 0.02 M rRNA nucleotides) . A further increase in formaldehyde concentration does not affect the rate of formation of methylene bridges (16). The concentrations of hydrogen ion and CH,O do not affect the secondary RNA reaction with formaldehyde in the same way as they do the secondary reactions of adenosine and purine mononucleotides (see Section 11, B ) . The elucidation of this fact requires more detailed study allowing, in particular, for the role played by the macrostructure of RNA in the reaction with CH,O. At 37°C the amount of methylene cross-links formed is 2.5 times higher than a t 20” (tRNA, p H 4.8) ( 5 6 ) . Dialysis does not reduce the amount of the cross-links (16). The effect of ionic strength and Mg2+is dealt with in Section 111, D, 2.

C. Deoxyribonucleic Acids I n 1953 Zamenhof, Alexander, and Leidy (58) reported that formaldehyde a t high concentrations ( 4 M , p H 7.2, 30”) causes a sharp decrease in the transforming activity of DNA and a gradual drop of viscosity. This does not happen with 0.33M CH,O. It was suggested that formaldehyde reacts with the amino groups of nucleic acid and destroys hydrogen bonds. Later, direct proof of the interaction of DNA with formaldehyde was presented (11).I t was shown that denatured DNA was capable of adding [14C]f~rmaldehyde(for the role of the secondary structure of DNA in this reaction, see Section 111, D, 3 ) . The reaction of DNA with formaldehyde is much less well understood than are the reactions with mono- and polyribonucleotides. However, it can be expected that the bases in DNA react with C H 2 0 in principle just as do the free bases or bases in RNA. Early results (11) on the RNA-formaldehyde reaction, which gives not only labile derivatives but also derivatives remaining intact after prolonged dialysis, were also found t o be valid for denatured DNA ( 5 9 ) . The DNA reaction with 14CH20was carried out under denaturation conditions (10 minutes, 100°C). A small part of bound radioactivity was not removed even on prolonged dialysis (up to 5 days) ( 5 9 ) . According to spectral evidence, the primary formaldehyde reactions with ribo- and deoxyribonucleoside phosphates result in the same (monomethylol) derivatives (see Section 11, A). Similar changes of ultraviolet spectra are observed after formaldehyde treatment of single-stranded and denatured double-stranded bacteriophage DNA’s (60, 8). The evidence for methylene bridge formation between purines, reviewed in Sections 11, B and 111, B, 2, suggest that a t least some of the so-called “firmly bound formaldehyde” that is not detached from DNA

20

M. YA. FELDMAN

on dialysis is actually in methylene bonds. Some authors tried to determine by ultracentrifugation whether DNA chains separate on formaldehyde treatment and thermal denaturation or are prevented from doing so by the cross-links formed. Centrifuging in a density gradient (61, 62, 69) gave equivocal results. Analytical ultracentrifuging appears to indicate the formation of cross-links hindering the complete separation of chains (6% 64) *

D. Effect of Secondary Structure Numerous studies have been concerned with the effect of the secondary polynucleotide structure on the reaction of formaldehyde and with the reverse relation, that of the effect of the reaction on the structure of the polymer. The evidence available can be summed up as follows. 1. The base functional groups involved in the formation of hydrogen bonds do not react with formaldehyde, and, conversely, bound formaldehyde hinders the formation of the usual hydrogen bonds between complementary bases of nucleic acid. 2. The stacking interaction of bases does not hinder the reaction of amino groups with formaldehyde. In turn, the reaction with CH,O does not prevent base stacking. 3. Formaldehyde does not destroy the polymer chain, does not disrupt base stacking, and does not seem by itself to break hydrogen bonds. It does, however, make nucleic acid more sensitive to the action of denaturing agents, in particular to the action of heat (the “melting temperature” of DNA is lowered in the presence of formaldehyde). This may be explained by the fact that in the presence of formaldehyde denaturation is irreversible because the formaldehyde bound to the bases hinders rmaturation. The main experimental evidence presented below is principally obtained a t the first reaction stages when practically only methylol derivatives are formed. (The effect of the secondary structure on the formation of methylene bridges has been studied only for ribonucleic acids; see Section 111,D, 2.) 1. SYNTHETIC POLYNUCLEOTIDES

The stable complex of poly(U) and poly(A) (0.1M phosphate buffer, room temperature) is similar to DNA in not binding [14C]for-* maldehyde (in 0.05% solution). However, when the ionic strength is low, (0.001M phosphate), poly (A) reacts with formaldehyde as if there were no poly(U) in the solution a t all (11). Thus the DNA-like secondary structure fixed by hydrogen bonds hinders the formaldehyde reaction with bases.

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

21

The question how formaldehyde treatment affects the polynucleotide secondary structure has been discussed by Haselkorn and Doty (6). Having studied differential spectra, the authors chose a wavelength a t which changes in the intensity of absorption characterize either the extent of denaturation or only that of the reaction. At about 280 nm for poly(A,U) and 290 nm for poly(I,C), the changes in optical density are due to formaldehyde binding, whereas at about 260 and 250 nm, respectively, they are due only to denaturation. Kinetic studies during the short incubation period (up to 1.5 hours) at pH 6.8 (0.11 M phosphate) showed formaldehyde to cause denaturation of polynucleotide complexes a t a relatively high concentration (about 1 M) and somewhat elevated temperature (35”C), the rate of denaturation greatly exceeding that of the reaction and, thus, not limiting the latter. Poly (A,U) reacts with formaldehyde a t the same rate as does poly(A). Similarly, poly(1,C) reacts a t the same rate as does poly(C). However denaturation becomes markedly slower as the ionic strength is raised to 1.0. Under these conditions, it limits the reaction and lowers the formaldehyde interaction rate with amino groups. It is to be noted that what is termed by Haselkorn and Doty (6) as “denaturation” has been actually shown by further studies to be a much more complex process, apparently involving thermal denaturation and a fast formaldehyde addition to uridine and inosine residues that cannot be registered spectrophotometrically. The formaldehyde added to ring -NH-COgroups hinders renaturation and thus favors the less rapid formaldehyde reaction with exocyclic amino groups reported by the authors. Such an interpretation is in accord with experimental data on polydeoxynucleotides, poly (dA,dT) and poly (dA) apoly (dT) (65). The alternative concept of the “induction effect” (66) (see Section 111, D, 3), which completely ignores the very fast CHzO addition to the ring NH-groups, can hardly provide a better understanding of the process. The effect of formaldehyde concentration on denaturation of polynucleotides was studied with poly(1) (6). When the concentration is increased by 1% (ionic strength of about 0.9, pH 6 ) , the T, drops by about 18”. To minimize denaturation it is proposed (6) to make use of as low a CH,O concentration as possible (about 0.1 M ) , high ionic strength and low temperature (about 20°C). It has been shown by optical rotatory dispersion (50) that in neutral solution poly(C) has a highly ordered secondary structure. It is not destroyed in the presence of formaldehyde, i.e., it contains few or no hydrogen bonds, and it is completely disrupted by ethylene glycol as would be expected to be the case for structures maintained by hydro-

22

M. TA. FELDMAN

phobic interactions. It is thought (50) that poly(C) a t pH 7 has a single-stranded helical structure stabilized by intrastrand stacking of pyrimidine bases. (The pH is critical as poly(C) a t pH 4.1 has a hydrogen-bonded, double-stranded structure.) The reaction with CH,O plays an important role for two reasons. First, it helps to elucidate the nature of forces maintaining the secondary structure of poly(C) in neutral medium, and second, it shows that base stacking does not affect the formaldehyde reaction with amino groups and is not destroyed by it. Formaldehyde treatment of poly(C) was carried out by heating up to 90°C followed by slow cooling down to 20" (50), guaranteeing the irreversible destruction of hydrogen bonds (see Section 111, D, 3 ) . Yet, the Cotton effect, whose intensity can be considered as a measure of asymmetry (helicity) of poly (C) molecules, is the same whether poly(C) is treated with formaldehyde or not. It is thus seen that the secondary structure of poly(C) a t pH 7 has no relevance to hydrogen bonding and CH,O does not destroy the forces maintaining this structure. This was also proved in the same work (50) by the following facts. a. Rising temperature affects the intensity of the Cotton effects of poly (C) and formaldehyde-treated poly (C) similarly. In both cases, the changes are gradual and noncooperative as distinct from the temperature dependence for the DNA-like double-stranded structures supported by hydrogen bonds. b. On formaldehyde treatment, the absorption spectrum of poly (C) undergoes the usual changes: the intensity of the maximum rises by 14% and the maximum is shifted to longer wavelengths by 4-5 nm, showing that the reaction actually takes place. However, on heating treated and untreated poly (C) , the relative increases in optical density due to thermal denaturation were both gradual and identical. I n the experiments with formaldehyde-treated poly (C) , structures stabilized by hydrogen bonds were certainly absent before thermal denaturation started. The hyperchromic e k c t was caused in this study by the disruption of other forces maintaining the secondary structure of poly (C) in neutral medium, such as, possibly, the stacking interaction. Fasman et al. (50) proved that formaldehyde does not affect the stacking interaction. Later, Stevens and Rosenfeld ($9) showed unequivocally that single-stranded base-stacking does not affect the chemical affinity of bases for formaldehyde. Their work on poly(A) a t p H 7.5 and with 1-2% CH,O suggests that: (i) a t p H 7.5, poly(A) has few or no hydrogen bonds; (ii) the secondary structure of poly(A) can be judged by the form of the thermal denaturation plots, which resemble those of poly(C) and are apparently due to stacking interaction between adjacent bases, with formaldehyde leaving the Structure intact; and (iii) base

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

23

stacking does not hinder the interaction of bases and formaldehyde as evidenced both by an almost quantitative addition of ["C] formaldehyde without any disruption of the secondary structure and by the results of comparative kinetic studies of formaldehyde reactions with poly (A) and adenosine. The ratios of the rate constants for the direct and back reactions were practically identical for the interaction of formaldehyde with poly(A) and in the reaction with adenosine at 30", 35", 40", and 45°C. The absolute values of these constants were somewhat lower in the former than in the latter case. 2. RIBONUCLEIC ACIDS At a low ionic strength, all the amino groups of tRNA can react with formaldehyde at 25°C. The extent of the primary reaction is determined by the equilibrium a t the particular concentration of reagents chosen and is independent of the initial secondary structure (9).As the ionic strength is raised, the rate and extent of both the primary (11, 57, 66) and secondary (16) reactions fall. Mg2+ions are particularly effective in this respect (Fig. 3). RNA is generally much less stable to formaldehyde than is doublestranded DNA. The hydrogen-bonded regions of RNA are much smaller and less stable than those of DNA, the cooperative effects being accordingly weaker. It might be suggested that limited and short-lived unwinding of double helices is much more frequent in RNA than it is in DNA, thus providing more opportunities for base interaction with CH,O. Formaldehyde addition to the double-helical regions of RNA thus becomes possible a t room temperature and a t low concentrations of CH,O. Stabilization of RNA double helices by means of Mg2+ gives them almost the same stability as DNA. At room temperature and in the presence of Mg2+,a high percentage of tRNA bases is completely unavailable to the action of formaldehyde [Fig. 3; see also references (67, S S ) ] . Similar results were reported for tRNAPheat 35°C (66). The influence of formaldehyde treatment on the hyperchromic effect (57, 32, 69) deserves special consideration. Figure 4 shows typical "melting curves" of RNA. With untreated RNA, the sigmoid curve reflects the cooperative process of thermal denaturation. As the ionic strength is decreased, the midpoint of the ascending part of the curve, the T,, is shifted toward lower temperatures. The gradual and comparatively small increase in absorption on heating RNA treated with formaldehyde is independent of the ionic strength. The shape of the curve resembles those of poly (A) and poly(C) (either treated or untreated with formaldehyde) in neutral solutions (50, 2 9 ) . This resemblance, as well as the absence of

24

M. YA. FELDMAN

F---l

0.55

10

30

50

70

90 OC

FIQ.4. Absorbancc as a function of temperature of TMV-RNA bcfore and after reaction with formaldehyde (32).RNA was treated with 1.2 M CH20 for 15 minutes at 85' and rapidly cooled. Measurements were made at 260 nm. At this wavelength, the changes in optical density with rising temperature depend on thermal denaturation only, rather than on the reaction of bases with formaldehyde (heating formaldehyde mixtures with mononucleotides has practically no effect on optical density a t 260 nm). Control (without CH20) in 0.1M phosphate buffer, pH 7.5 ( 0 ) and 0.001M phosphate (0) ; RNA treated with formaldehyde in 0.1 M phosphate (A) and 0.Wl M phosphate ( A ) , Similar curves for thermal denaturation were obtained in experiments with other virus RNA's (32, 70) and with rRNA (71).

the ionic strength effect, shows that hypochromism and, accordingly, the secondary structure of RNA treated with formaldehyde are due to the interaction of stacked bases rather than to hydrogen bonds. I n such an RNA there are few or no double-stranded helical regions. The change in the absorbance of hydroxymethylated RNA on heating is reversible and is eliminated on cooling (32,68-71). A record of the dependence of optical density on temperature, not after RNA treatment with formaldehyde but from the beginning of this treatment, gives the usual melting curves showing a sharp increase in optical density over a limited temperature range. I n this case, cooling reveals two hypochromic fractions. The first, due to base stacking, is rather small and reversible, the second is large and irreversible (70,7 1 ) . Formaldehyde addition to RNA amino groups makes it impossible for

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

25

them to participate in the formation of hydrogen bonds (57) but does not hinder base stacking (68, 69, 32) under suitable temperature. Melting curves similar to those presented in Fig. 4 have been recorded by Hall and Doty [see Fig. 2 in reference ( 5 7 ) ] ,who used urea rather than formaldehyde as ti denaturant. It was later shown that urea fails to give such unequivocal effects as a denaturing agent as does CH,O (72, 7.3, because it appears to weaken rather than to completely abolish both hydrophobic interactions (74) and hydrogen bonding (72). Formaldehyde treatment increases the effective hydrodynamic volume of tRNA molecules. The sedimentation coefficient of tRNA falls by 0.40.7 Svedberg unit (69, 56). Thc intrinsic viscosity rises from 0.06 dl/g to 0 . 0 9 4 1 dl/g (69) and remains so even after free formaldehyde is removed (tRNA precipitation with ethanol and dialysis for 18 hours) (69). A more prolonged dialysis (for 48 hours), to remove not only the free but also the labile bound formaldehyde, results in almost complete restoration of the initial tRNA sedimentation rate. The presence of methylene bonds does not affect the sedimentation characteristics of tRNA ( 5 6 ) . The sedimentation and chromatographic results point to no chain rupture or dimerization (or polymerization) of tRNA on prolonged formaldehyde treatment (15 days, 20°C, pH 4.8). After limited guaniloribonuclease digestion of tRNA containing methylene bridges, large fragments (“halves”) are not separable from each other. Thc methylene cross-links formed both in the presence and the absence of Mg2+ are intramolecular, of the type

TCH7 . . R-

-It.

rather than intermolecular (66). The reversible decrease of Sedimentation coefficients without rupture of the chain on CH,O treatment is also observed in experiments with ribosomal and virus RNA (75-78). The aggregation of RNA a t pH <4.2 and low Mg2+ concentration leads, in the presence of 7.7% CH,O (20 minutes, 60°C), to a quantitative formation of stable specific dimers (78).The 28 S molecules combine with 28 S rRNA only. Similar specific complexes are formed by the 16 S rRNA of E. coli and by the RNA of bacteriophage MS2. Thus formaldehyde appears to fix the dimers by means of interchain cross-links (78, 79):

-RI

CHI

I

-R-

26

M. YA. FELDMAN

3. DEOXYRIBONUCLEIC ACIDS

Native double-stranded DNA fails to react with formaldehyde (6,11). For reaction to occur, it is necessary to destroy the hydrogen bonds of DNA by prolonged dialysis against watcr (11, 80),heating, or alkaline treatment (81). I n some cases, particularly in experiments with heating, the denaturant and formaldehyde are both used, but the mechanism remains the same as whcn the denaturant acts before the formaldehyde. After reaction with formaldehyde, DNA is not capable of renaturation (8, 61). The effect of formaldehyde on the secondary DNA structure is shown by thc melting curves in Fig. 5 (82). I n the absence of formaldehyde and on slow cooling, the DNA is almost completely renatured. On repeated heating, DNA “melts” as usual, the optical density increasing sharply a t about 82°C. The picture is different in the presence of formaldehyde. At first, denaturation proceeds as sharply as when no formaldehyde is present (this part of the curve is not shown in Fig. 5 ) , but a t a lower temperature. At 1% CH,O, the T,,,decreases by about 15°C. A stronger ‘Lhyperchromic” effect (Fig. 5 ) is due to the modification of chromophoric groups. When the temperature is lowered, no renaturation takes place. A small decrease in optical density does not account for even partial renaturation, as evidenced by the form of the curve, the optical density changing gradually both on cooling and repeated heating. However, the presence of some hypochromism points to a secondary structure of hydroxymethylated DNA. On repeated heating, the temperaturedependence curve resembles those for poly(A) and poly(C) in neutral

I

1

1

25”1Oo0 80

I

1

60

40

IIII

I

20”20 40 Temperature ( C ” )

1

I

I

60

80

100

FIG.5. Changes in absorbance at 260 nm on hcnling, slow cooling, and reheating T4 bacteriophage DNA with and without formaldehyde (6’2) (pH 8, 0.37 M CH20). 0,+HCHO; 0 , -HCHO. By permission of Academic Press, Inc., New York.

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

27

medium both in the presence and the absence of CH,O as well as that for hydroxymethylated RNA. This likeness suggests that hydroxymethylated DNA has a secondary structure maintained by base stacking only. Such a structure is not peculiar to native DNA, being due to formaldehyde treatment (this holds only partly for RNA, and the secondary structure of poly(C) and poly(A) at pH 7 is unaffected by formaldehyde; see Sections 111,D, 1 and 2 ) . There is enough evidence to justify the presumption that on partial denaturation caused, e.g., by moderate heating, only unwound sites of deoxyribonucleic acid react with formaldehyde. As formaldehyde prevents renaturation, the cooling of the solution leaves these sites open. It is possible to find by electron microscopy the DNA regions denatured by heating and fixed with formaldehyde (83,84). These are distributed in DNA in a strictly regular fashion rather than randomly. Their number, size and location in the molecules of a particular deoxyribonucleic acid are quite reproducible. Thus, these sites have a specific structure. I n the presence of formaldehyde, DNA undergoes denaturation just as it does in its absence, with the destruction of A - T pairs preceding that of G - C pairs. This fact was proved by two methods, differential spectrophotometry, which allows the melting of A . T and G - C pairs of DNA to be observed separately (85), and by electron microscopy (86). The latter is used to follow the partial denaturation of deoxyribonucleic acids with different base compositions. DNA is heated in 12% CH,O solution a t various temperatures (46"-52") for 10 minutes and then quickly cooled. I n the DNA molecules of papilloma virus with a high content of A - T pairs ( 5 8 % ) , the denatured sites arc evident a t lower temperatures than in those of polyoma virus with fewer A * T pairs (52%).The DNA molecules of Cancer pagurus with very high content of A - T pairs (over 90%) and the synthetic polymer, poly (dA,dT) , melt a t relatively low temperatures following the principle "total or no" change. No melted sites are to bc seen in these polynucleotides. Thus, in the presence of formaldehyde, DNA starts melting in regions enriched with A - T pairs, just as it docs in its absence. Therefore, formaldehyde acts to fix denaturation changes in the DNA structure caused by thermal fluctuations (85). This conclusion is not coiitradictcd by the facts that in the presencc of formaldehyde the melting temperature of DNA is lowered and its denaturation is dependent not only on tcmperaturc, but on CH,O concentration as well. The structure of DNA is dynamic rather than static, being determined by the cquilibrium between two processes: denaturation and renaturation (87, 65, 88). This equilibrium dcpcnds on temperature, with higher temperatures shifting it toward denaturation. I n the presence of formalde-

28

M. YA. FELDMAN

hyde, denaturation becomes irreversible and, therefore, the strand separation takes place a t a lower temperature, This seems to account for the mechanism of the T, lowering of DNA in the presence of formaldehyde (8, 63, 87). DNA denaturation is dependent on the concentration of CH,O. At 45”C, pH 7.2 and an ionic strength of about 0.1, 1% formaldehyde was practically unreactive with phage T4 DNA (no spectral changes observed during 48 hours), but reaction did take place in 15% CH,O solution (81). I n view of the above facts, the effect of high CH,O concentrations can be considered as due to an acceleration of formaldehyde reaction with opening sites produced by thermal fluctuations rather than to an active denaturation of DNA. Thus with respect to DNA, formaldehyde is not an “active,” but a “passive,” denaturing agent that opposes renaturation without causing denaturation. The reaction proceeds only with denatured DNA or with its denatured sites. A pn’ori, it is anticipated that the sites a t the ends of helices (where the chain is ruptured, a t the ends of molecules, or at sites adjacent to the already unwound DNA fragments in which the separation of chains is fixed by formaldehyde) should be less stable on heating than those in the middle of helices (89, 90, 65). Hence, formation of fixed centers of unwinding on moderate heating of DNA in CH,O solutions would induce the accelerated thermal denaturation of adjacent sites (“induction effect” (65)). Such a picture is in accordance with the results of detailed kinetic investigations (81, 89,90,65). However, the number of denatured nucleotide pairs of bacteriophage T 7 DNA containing free (with no CH,O added) amino groups is found to be very small (spectroscopy a t two wavelengths, 3.7% CH,O, pH 9.1, 58°C) ( 6 5 ) .It is doubtful that the induction effect plays an important part in the DNA interaction with formaldehyde. The cooperative character of the helix-coil transition is practically completely retained (the presence of formaldehyde in the solution does not affect the width of the DNA melting range). Electron microscopy (91) does not provide reliable evidence on accelerated melting of the ends of helices.

E. Effect of Formaldehyde on the Functional Activity of Nucleic Acids Some interesting results have been reported on the effects of CH,O modification of RNA’s on their functional properties. It was most surprising to find that under certain conditions modified RNA’s retain a considerable part of the original functional activity, sometimes even in greater degree.

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

29

The amino-acid accepting ability of transfer RNA is completely or almost completely suppressed by omission of Mg2’ from the reaction medium (92-94, 56). However, tRNA that has reacted with CH,O in the presencc of Mg2+ ions retains a considerable amount of its accepting activity. The activity of unfractionated tRNA with respect to four amino acids decreased by 30-50% on treatment with 3% CH,O (pH 7.6, 25°C) during the first 10 hours. Inactivation then stopped completely (92, 93) while the formaldehyde reaction continued. The primary reaction with amino groups is over in 100 hours when 15% of the NH,-groups has been converted to -NH-CH,OH-groups (9). Considerable retention of the acceptor activity in the presence of Mg2’ is also observed with a more intensive modification of tRNA leading to the formation of methylene bonds (1-4 per molecule) (56) whereas with no Mg2+the formation of 4 methylene bonds results in the total loss of acceptor activity (in this study hydroxymethyl groups were all or almost all removed by prolonged dialysis). Denaturation of tRNA in a formaldehyde solution not containing Mgz+(Section 111,D, 2) leads to a complete loss of the amino-acid accepting ability. However, an average of about 50% of the tRNA loses activity w e n with Mg2’ ions present in the solution (92, 93, 56). It is tempting to suggest (56) that intramolecular methylene bonds fix, in the presence of Mg“, not one, but at least two tRNA conformations, the “active” and “nonactive” ones, on the assumption that the two conformations were in equilibrium before formaldehyde fixation. This hypothesis is in accord with the results obtained when studying the acceptor activity of the yeast tRNAV”’ modified by CME-carbodiimide (95).3 Similar ideas are advanced with respect to chymotrypsin cross-linked with formaldehyde (96). The kinetics of CH,O inactivation of the “transfer” function of tRNA (as determined by the incorporation of [“C] phenylalanine into protein in the presence of poly(U) in a cell-free system) is rather similar t o that of the inhibition of acceptor activity. In this study (93), the formaldehyde treatment of the tRNA (an unfractionated preparation from Escherichia coli) was carried out over periods of time in which only methylol, but not methylene, derivatives would be expected to be formed. Some interesting results on the action of formaldehyde on mRNA were obtained for RNA bacteriophage f2 (97), whose messenger activity in the cell-free system is enhanced by mild treatment with 1 M CH,O a t 37°C for 11 minutes (followed by cooling and ethanol precipitation). Such a pretreatment of f2 RNA intensifies the synthesis of four proteins specific to this viral RNA and induces the synthesis of a t least three more CME-carbodiimide is N-cyclohexyl-N’-/3-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate.

30

M, YA. FELDMAN

polypeptides. All syntheses begin with N-formylmethionine, being thus initiated by AUG or GUG codons. By fixing the denaturation of the RNA a t limited sites, formaldehyde seems to make these codons more available for ribosomes thus leading either to increased synthesis of usual proteins or to the appearance of new polypeptides, depending on the localization of these codons. The CH,O modification has thus enabled us to obtain proof for the important functional role of the conformation of tRNA and mRNA. The same modification has also been used to prove the dominant role of the primary rather than secondary structure. Ribosomal RNA (from rabbit reticulocytes) was treated with 3% CHzO a t 63°C for 15 minutes (with subsequent cooling) to destroy completely the secondary structure (98). It was found that the resulting rRNA with disrupted secondary structure is hydrolyzed by ribonuclease T1 (in the same 3% CH,O solution) much more rapidly than is the normal rRNA. The intermediate products of partial ribonuclease (Tl) hydrolysis (10 minutes, 0°C) were discovered, however, to be similar as far as their molecular weight is concerned both for the native and formaldehyde-treated preparation of rRNA. Thus the disruption of the secondary structure does not affect the points of attack of the enzyme. Hence ribonuclease T1 recognizes definite nucleotide sequences. These experiments have excluded an alternative explanation. Formation of relatively stable intermediate products cannot result from the existence of “weak” sites (more available to the enzyme) in the RNA secondary and tertiary structures. This is because these products are also formed when the enzyme acts upon the fully unwound RNA molecules.

IV. Interaction of Formaldehyde with Nucleoproteins

This interaction involves formaldehyde reactions with nucleic acid and protein. It might well be that nucleoproteins undergo specific reactions with formaldehyde to form bridges between the nucleic and protein moieties (93-101). However, no strict chemical proof for this hypothesis has as yet been presented. Before proceeding to review the results available on the action of formaldehyde on nucleoproteins, a brief look a t the present state of the problem of formaldehyde interaction with proteins is pertinent.

A. Formation of Methylene Bridges in the Reaction of Protein with Formaldehyde The main events in the reaction of protein with formaldehyde (under conditions close to physiological ones) can be depicted by the following scheme:

31

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

RNH, RNH-CH2OH

+ CH2O RNH-CH?OH + R'H + RNH-CH2-R' + HzO

At high formaldehyde concentrations, dimethylol derivatives, RN(CH,OH),, are also formed. No labile mono- and dimethylol derivatives, formed in very fast reactions, have been isolated; their identification was made indirectly by physicochemical investigations of amino acids in formaldehydc solutions (102,103). The results of thcsc studies were generalized in the classical revicw by French and Edsall (104) and were subsequently substantiated kinetically (105).Spectral evidence was also provided for the addition of formaldehyde to imidazole protein groups (a-chymotrypsin) (106). Slow secondary reactions result in stable condensation products, methylene derivatives (RNH-CH,-R') . The first to present proof for condensation in reaction of protein with formaldehyde were Nitschmann and Hadorn (107),who showed that when gaseous formaldehyde binds to casein, water is eliminated. However, such a condensation could supposedly take place also on formation of compounds R=CH, and again on formation of methylene bridges . (R-CHZ-R') The first alternative was disproved. Various physical approaches showed that the action of formaldehyde on proteins, under conditions close to physiological ones, gives intermolecular cross-links causing increased molecular weight of proteins and/or intramolecular cross-links hindering denaturation of protein and favoring its renaturation (108112). It was also found that no cross-links are formed when formaldehyde acts on proteins with acetylated amino groups (113,11.6, 108). Methylene cross-links are most likely formed between amino groups and those of amide, guanidyl, phenol, imidazole or indole (114, 115). This conclusion was based on the study of a number of model systems such as alanine CH,O acetamide, proline + CH,O acetamide, threonine + CH,O + $,Cdimethylphenol, threonine CH,O + a-N-acetyl-L-histidine, polyglutamine C H 2 0 alanine, methylene tyrosine polymer CH,O methyl guanidine, etc. The first three reactions yield crystalline products (114, 116) and other products not isolated but. identified by analytical tests, in particular, by color reactions. I n the first stage, formaldehyde is added to the amino group to form the aminomethylol derivative (114) whose secondary reaction with the primary amide or another functional group givcs rise to compounds of the type

+

+

+

+

RNH-CH,-NHCOR',

+

+

RNH-CI&-NHC<

+

+

/m NHR'

As distinct from primary amides, secondary amides do not undergo

32

M. YA. FELDMAN

condensation with formaldehyde and amines under similar conditions (weak acid, room temperature) (114). The claim made a priOri by Nitschmann (107,113)about the formation of methylene bridges between c-amino groups of lysine residues and peptide bonds in adjacent protein chains was actually rejected in model investigations by Fraenkel-Conrat and Olcott. Special investigations were also made of the possible formation of methylene bridges between amide and guanidyl groups as well as between phenolic and amide or phenolic and guanidyl groups (114, 116). These alternatives could not be confirmed. Attempts to obtain methylene bridges between primary and secondary amide groups and between amides and aromatic rings failed even with acid used as catalyst (117). There appears to exist no convincing evidence for the formation of methylene cross-links between the amino groups of protein (114, 108). Direct proof was recently presented for methylene bridge formation by the action of formaldehyde on proteins (118, 119). Formaldehyde reacts with a-N-acetyllysine and a-N-acetyltyrosine to give, a t 37"C, a condensation product that, after deacetylation ( 1 M HC1, 37"C, 48 hours), leads to compound VII ( Lys-CHn-Tyr).

In the same studies, this coinpound was isolated from hydrolyzates of proteins treated with formaldehyde, that is, from serum albumin and toxoids of tetanus and diphtheria. Many authors have determined the amount of formaldehyde bound to proteins (120,121). Of greatest interest appear to be estimations obtained with labeled [ 14C]formaldehyde (122).Under conditions used to prepare toxoids (0.033 M CIZO, or 2200 moles of formaldehyde per mole of diphtheria toxin, a t 37°C) the protein reactions are complete in 15 days. The formaldehyde bound is distributed as follows: part is bound reversibly and can be removed by dialysis; 1 mole of toxin (MW 64,500) binds irreversibly 62 moles of formaldehyde, 14 of which are not liberated even by acid hydrolysis. Lys-CH2-Tyr, amounting to 4 moles per mole of anatoxin, is also stable to acid. Sedimentation constants of the diphtheria and

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

33

tetanus toxoids show little or no difference from those of the starting toxins (about 4.2 S for all cases studied) (123-125). A number of formaldehyde reactions with amino acids and proteins that lie outside the scope of this review have also been reported. Among these are reactions characteristic of free amino acids rather than of proteins, and protein reactions proceeding under conditions that are very far from physiological ones (in the presence of acid or alkali, or heating). Thesc reactions arc reviewed in special works on chemistry of amino acids, proteins and formaldehyde (10.4, 126-128, 1.4).

B. Effect of Formaldehyde on Nucleoproteins 5-Hydroxymethyluracil, obtained from the addition of formaldehyde to uracil (see Section 11, A, l ) , undergoes condensation with some amino acids (26) yielding products in which the residues of uracil and of amino acid are linked by methylenc group. The amino acid residue is connected to the mcthylene group through a sulfur atom (cysteine, homocysteine) or through an amino group (glycine). The reaction proceeds in the presence of acid or alkali and a t elevated temperature, that is, under conditions that are very far from those used with nucleoproteins. More adequate low molecular models do not appear in the literature. The results available on formaldehyde action on nucleoprotein particles such as ribosomes indicate that nucleoprotein fixation is the main result of this effect.

1. RIBONUCLEOPROTEINS There is a resemblance between the changes in ultraviolet spectra of ribosomes and in those of free RNA on formaldehyde treatment (129, 130). These spectral changes serve as proof of formaldehyde addition to the amino groups of rRNA bases in ribosomes. Just as with free rRNA, of ribosomes decreases in the presence of formaldehyde. The dethe T,,, crease is the greater, the stronger the formaldehyde concentration (130). The thermal denaturation of double helices of rRNA, both free and bound in the ribosome, is irreversible in the presence of formaldehyde (the starting double-helical structures are not reestablished on cooling) (130). Such a denaturation of RNA in the ribosome does not, surprisingly, change the hydrodynamic volume of the ribosome particle. Viscosity (130) and sedimentation coefficients of ribosomes (1.29) remain unaffected. It is to be noted that, in these viscosity and sedimentation studies, formaldehyde was not removed from the solutions (lSO),or the removal was effected only partially (129). Under such conditions, the free RNA has an increased effective hydrodynamic volume (Section 111, D, 2). Conversely, thermal denaturation of ribosomes in solution, with no

34

M. YA. FELDMAN

formaldehyde added, causes changes in ribosome conformations that are not fully reversible, judged from the same viscosimetric and sedimentation approaches (131). Ribosome fixation by formaldehyde is apparently due to methylene bonds formed essentially in the protein part of the nucleoprotein (129, 130). Neither high ionic strength (132) nor the presence of dodecyl sulfate (129) or urea (101) induces protein separation from RNA when ribosomes are given a preliminary treatment with formaldehyde. For ribosomes to be fixed, a very mild formaldehyde treatment suffices. Thus, for example, treatment of ribosomes with 0,05% CH,O a t 20°C and p H 7.5 for 75-120 minutes prevents their dissociation to proteins and RNA under the action of 1% dodecyl sulfate. The same treatment a t 0" is insufficient (129). I n other cases short incubation with 24% formaldehyde gives, a t a low temperature (0"4"C,1 hour, p H 6),the necessary fixation and proves useful in electron microscope studies (133, 134) and on ribosome centrifugation in the CsCl density gradient (132). The explanation of the fixing effect of formaldehyde under such mild conditions meets with considerable difficulties. Short treatment periods, low temperatures and neutral rather than weak acidic media do not favor the formation of methylene cross-links, as is known from the results of formaldehyde reactions with proteins and nucleic acids. It may be supposed that stabilization of nucleoproteins necessitates very few crosslinks. It is also likely that the nucleoprotein particle, having a rather rigid structure (e.g., the ribosome), involves functional groups held in a position sterically favorable to their linkage by means of methylene crosslinks in the reaction with CH,O. This steric factor may cause a sharp acceleration of the reaction. 2. DEOXYRIBONUCLEOPROTEINS The first to draw the attention to the interaction of nucleohistone with formaldehyde were Romakov and Bozhko (135). Later, the properties of the product of this reaction were studied in detail (100). Nucleohistone (of chromatin from pea buds) was treated with formaldehyde under extremely mild conditions (24 hours, O'C, p H 7.8), and formaldehyde was then removed by dialysis (100).Under these conditions, 0.5-1% CHIO concentration is enough to fix the nucleoprotein complex. I n contrast to native nucleoprotein, such a complex does not liberate protein in a saline solution of high ionic strength and can be studied in the CsCl gradient. It was found that its buoyant density is 1.411 g/ml, the same as the value calculated for the DNA-protein ratio in nucleohistone. Nucleohistone treated with formaldehyde is more stable to heating than is the native material, and the T, of the treated nucleohistone is

REACTIONS OF FORMALDEHYDE WlTH NUCLEIC ACIDS

35

higher than the T , of the untreated complex. With ribosomes, an opposite relation was observed (see above). Such a divergence in T , might be due to the different experimental conditions used: the T , of ribosomes was determined in the presence of formaldehyde, that of nucleohistone was estimated after CH,O was removed by dialysis. Even after the fifth treatment with Pronase, not all protein is removed from nucleohistone treated with formaldehyde; 4% of the protein remains firmly bound to DNA. I t is suggested (100) that formaldehyde reacts with nucleohistone to form methylene bridges between nucleic acid and protein.

V. Use of Reactions of Nucleic Acids and Nucleoproteins with Formaldehyde

No reaction of nucleic acids seems to have been as useful in experiment and practice as that with formaldehyde. The main fields of its application are the following: (1) study of nucleic acids and nucleoproteins, their structure and biochemical functions; (2) fixation of various biological structures, from ribosomes and bacteriophages to microscopic tissue sections; (3) inactivation of viruses for producing vaccines; and (4) chemical mutagenesis. The first three applications are essentially based on the fixing action of formaldehyde. This effect involves two mechanisms and, respectively, leads to two main results. Hydroxymethyl groups formed in the primary reaction of nucleic acid with formaldehyde fix denaturation by hindering renaturation. Methylene cross-links, formed in the secondary reaction under conditions not causing denaturation, fix the structure of the native RNA or of nucleoprotein. The type of fixation used depends on the particular aim pursued in the work. Of all applications of the reaction not concerned with the fixing action of formaldehyde, the most important is chemical mutagenesis. Aldehyde fixation in morphological studies does not receive special consideration in this review. The fixation of nucleoprotein particles and complexes of RNA’s has been discussed in Sections IV, B and 111, D, 2. The biochemical basis for the application of aldehydes (formaldehyde and acrolein) together with metachromatic dyes for histochemical investigations of RNA and DNA is dealt with in references (136-138).

A. Structural and Functional Studies of Nucleic Acids Formaldehyde is often used when it is desired to destroy double helices and to prevent their regeneration throughout the whole experiment.

36

M. YA. FELDMAN

Denaturation is usually attained by raising the temperature in the presence of formaldehyde followed by rapid cooling of the mixture, heating causing denaturation and formaldehyde preventing renaturation. Formaldehyde possesses a number of advantages over other denaturing agents: the necessary effect is produced by low concentrations of formaldehyde, nucleic acid can be investigated in an essentially aqueous medium, methylene glycol (hydrated formaldehyde) does not absorb ultraviolet light. Some important applications of “fixed” denaturation have already been considered above, namely, the use of formaldehyde reactions in functional studies of nucleic acids [Section 111, E; see also ( 1 3 9 ) ] , denaturation of polynucleotides in the presence of formaldehyde to study base stacking in poly(C), poly(A) and RNA (Section 111, D ) , location by means of electron microscopy of sites of DNA molecules enriched by A.T pairs [Section 111, D, 3; see also (91, 1.40,1 4 1 ) I . The primary formaldehyde reaction is of great value to achieve the separation of polynucleotide chains in order to elucidate their actual size. Thus, for example, heating the giant (>26 S) “DNA-like” RNA of yeasts in the presence of formaldehyde has showed that the preparation consisted actually of aggregates involving RNA molecules with molecular weight close to that of the 17 S component of rRNA (142). The same method has made it possible t o determine (81) that the polynucleotide DNA chain of phage T 4 is continuous and that its molecular weight is half that of the native DNA. This has provided one of the most convincing proofs that the DNA molecule consists of only two uninterrupted polynucleotide chains. Formaldehyde reactions also used for many other purposes, in particular when it is necessary to distinguish single- and double-stranded virus nucleic acids (60, 70, 143), to determine quantitatively the double-helical content of RNA (6, 32) and the number of secondary structure “defects” to eliminate specific features in the conformation of in DNA (89, a), some ribonucleic acids for a more exact estimation of their’ molecular weight (76, 77, 7 2 ) ,to separate the DNA chains in immunological experiments (82, 144), to study nucleic acids in situ in bacteriophages (146147) and in cells (136, 137).

B. Inactivation of Viruses by Formaldehyde in Vaccine Production

Formaldehyde action on virus nucleoproteins is of great practical value for preparation of viral vaccines for human subjects (14.8) and animals (149). Many investigations have dealt with virus “formol” in-

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

37

activation [see, e.g. ( 1 5 0 ) ] , but the chemical mechanism of this process has not been studied directly. The following four features of formaldehyde interaction with proteins, nucleic acids, and nucleoproteins can be considered as the most essential for the CH,O inactivation of viruses in vaccine preparation. 1. Formaldehyde acts both on the protein and nucleic virus component without causing any destruction of polypeptide or polynucleotide chains. 2. Modification of bases, formation of cross-links in nucleic acid and “hardening” of protein ensure that the virus is reliably rendered harmless. 3. Formaldehyde fixes the conformation of nucleoprotein. Recently evidence was produced concerning the special role of antigen conformation in inducing the synthesis of antibodies (151). Most antibodies induced by TMV are likely to be specific for the structure of the surface of the virus particle, for its conformation, rather than for the amino-acid sequence of virus subunits (152). It has also been shown (153) that the nucleoprotein and the free protein of the turnip yellow mosaic virus (crystallizable preparations isolated from the plant sap) are identically precipitated by antiserum to nucleoprotein but, when injected into rabbits, induce the formation of antibodies differently ; the antigenicity of the nucleoprotein is much higher than that of the free protein. Thus, in order for antigenicity to be stabilized it is desirable to fix the nucleoprotein as such, not its protein only. This condition is met by formaldehyde. 4. The small size of the formaldehyde molecule facilitates its passage through the protein shell of the virus. The importance of this fact can be exemplified as follows. Kethoxal (a-keto-P-ethoxybutyraldehyde) inactivates the isolated TMV-RNA much more readily than does formaldehyde. On the other hand, RNA in the virus is inactivated by kethoxal more slowly than by formaldehyde. It might be suggested that the relatively large size of the kethoxal molecule hinders its passage through the protein shell (154). All these peculiarities of formaldehyde action provide a post-facturn explanation of the choice of formaldehyde as an inactivator and fixative for vaccine preparation. The future will tell whether this delayed theoretical explanation will help to improve the preparation of vaccines and to prepare new vaccincs. It is to be noted that soiiie L‘anoinalics”of formaldehyde inactivation are also being explained. This is the ease for the reversible inactivation of TMV (155) and poliovirus (156) on short-term formaldehyde treatment (apparently due to the formation of only methylol derivatives) and the increased stability of the carcinogenic virus SV40 to the inactivating

38

M. T A . FELDMAN

action of formaldehyde [ thc virus contains a double-helical DNA (149)1. The question is now poscd whether all viruses retain their conformation in the formaldehyde reaction. This question, as well as many others cannot be answered a priori. If formaldehyde inactivation fails to give a satisfactory immunological effect, special physicochcmical studirs with purificd virus prcparations will clearly be nccdcd.

C. Effect of Formaldehyde on the Genetic Apparatus of the Cell The mutagenic action of formaldehyde was first described by Rapoport in 1946 ( 3 ) . Of mutagens acting on multicellular organisms, formaldchyde is one of the most interesting and well known (157, 4 ) . Formaldehyde causes mutation in viruses (158, 159), bacteria (1601, Neurospora (161) and Drosophila [on injection into adult flies (162) or introduction into culture medium for larvae (3, 163, 13,10,4) 1. The mechanism of mutagenesis induced by formaldehyde is best known for Drosophila larvae. Formaldehyde causes mutation in male larvae only (163, 4) and solely a t a definite stage of spermatogenesis (immediately preceding meiosis) (164). This shows formaldehyde to be a highly specific mutagen ( 4 ) . Its mutagenic effect has something to do with DNA rcplication, bccause after DNA synthesis is over formaldehyde loses its activity and cxcrts no mutagenic effect on late spermatocytes (165). Formaldehyde can cause mutation (recessive lethal) if the culture medium contains AMP (13). Adenylic acid can be replaced by adenosine or RNA, but not by adenine, cytosine or inosinic acid. Deoxyadenylic acid is less effective than adenylic acid (13, 166, 167, 10, 157). Mutations appear to be induced in Drosophila larvae not by formaldehyde as such, but by its reaction product with adenylic acid. It is suggested (13, 10) that mutation is causcd by the methylene or hydroxymethyl dcrivativcs cntcring one of the DNA (or RNA ( 1 5 7 ) ) chains during synthesis. It should bc noted that participation of a very labile hydroxymethyl product in formaldchydc-induced mutagenesis appears to be unlikely. In any case, the mutagenic action of formaldehyde (on Drosophila larvae) is not a dircct and primary one, but a much more complex phenomenon occurring probably during thc replication of the DNA. I n Drosophila larvac, formaldchydc induccs crossing-over either in the presencc or abscncc of AMP. The second alternative is of considerable interest in principlc as the agents inducing crossing-over act also as mutagens (157, 168). I n this case, formaldehyde is of great value because it allows crossing-over to be induced under conditions (absence of AMP) preventing mutagenesis (157).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

39

Formaldehyde and number of its addition products, such as methylol derivatives of succinimide, glutarimide, piperidine, pyrrolidine, morpholine, dikctopipcrazine, 2-mercaptoiinidazolc, etc., arrest the growth of the mouse ascitic carcinoma in vivo ( 2 ) and lead to considerable changes in tumor metabolism. Expcrimcnts with mcthylene bis-guanylic acid give some indications as to the mechanism of formaldehyde effect (43). This compound, produced from the reaction of GMP and CH,O (see Section 11, B, 2), suppresses drastically the mitotic activity of human amniotic cells in continuous culture (strain FL). I n the presence of CH,(Gp),, DNA Synthesis is a t first enhanced for 1.5 hours (presumably owing to increased nucleotide pool in the cell) and then hindered. I n contrast, the synthesis of cell RNA is little affected. Reproduction of viruses containing RNA undergoes quite independent changes. With CH, (Gp), in the culture medium, reproduction of poliovirus in FL cells is first suppressed (for 8 hours) and then restored. A hypothesis has been advanced to account for these results ( 4 3 ) . After suitable enzymatic conversions in the ribosephosphate part, the methylene dinucleotide enters the newly synthesized DNA chain with one of its nucleotide residues in place of the appropriate normal nucleotide. During the next replication the free methylene dinucleotide residue can enter the complementary chain. Thus two DNA chains are bonded by a methylene cross-link -Piir1

-Piir-

and mitosis is inhibited. It is supposed that similar cross-links between the complementary chains of the replicative virus RNA inhibit the reproduction of the virus. The methylene bridge links two purine residues, i.e., a purine in one chain is opposed by a purine in the complementary chain, but not by pyrimidine as is usually the case. If the methylene cross-link is removed by hydrolysis or enzymatic reparation, chains will be separated. Purine substitution for pyrimidine thus causes mutation. This hypothesis would be substantially confirmed if methylene dinucleotide could be isolated from DNA. There are, however, as yet no suitable methods available to obtain such compounds from DNA. The highly specific mutagenesis induced by formaldehyde in Drosophila larvae cannot be even hypothetically accounted for (165, 4 ) . The results available (165,4 ) on localization of mutations (X chromosome and the second, but not the Y, chromosome) fail to explain why the formaldehyde reaction product with AMP affects the early spermatocytes

40

M. YA. FELDMAN

and why spermatogonia and all stages of oogenesis are not sensitive to the mutagenic activity of this reaction product. With [ ''C] formaldehyde in the culture medium, the label is discovered autoradiographically in the germ cells a t all stages (169).However, it is extremely doubtful whether this label always belongs to the product of the reaction between AMP and l'CH,O (tentatively CH, (Ap) ?). Just what cells this product permeates and how long it stays in different cells still remains unknown. It is possible that the carly spermatocyte has the most favorable conditions for accumulating methylene dinucleotide up to the concentration needed for its inclusion into the polynucleotide. The cytostatic action and cross-over induction do not necessarily require the intermediate formation of methylene dinucleotide. These effects may also be due to another mechanism such as the direct formaldehydc interaction with cell DNA.

VI. Related Reactions and Their Effects (as Compared to Formaldehyde Reactions) A detailed review of these reactions is not within the scope of this essay, particularly as they have already been dealt with in many reviews (38,170-17s). The purpose of this section is to show the place of formaldehyde as a modifying agent among other aldehydes and bifunctional compounds whose action on nucleic acids has been studied rather thoroughly. A. Miscellaneous Aldehydes A number of aldehydes other than formaldehyde are useful for modifi-

cation of nucleic acids. These are almost all dicarbonyl compounds. Their interaction with nucleic acid or its component is likely to involve two consecutive reactions with two carbonyl groups. Yet, only the stable end products of these reactions have been well studied; these are discussed below. Particularly well known are the reactions with 1,2-dicarbonyl compounds (154, 174, 176), namely, with glyoxal (OHC-CHO), kethoxal (CH,-CH (OC2H,)-CO-CHO) and pyruvaldehyde (CH,-CO-CHO) . These modifying agents differ from formaldehyde in the following respects. 1. They are more specific than formaldehyde, and stable condensation products are found only in their reactions with guanine, guanosine and deoxyguanosine. At the nucleic acid level, this specificity for guanine rcsiducs of RNA has been unequivocally shown only for kethoxal (176).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

41

(Vm) 2. l12-l)icarbonyl compounds do not form bridges between two niolccules (or residues) of the base. Two carbonyl groups react with two nitrogen atoms (N-1 and N-2) of guanine, forming a new cycle. I n formula VIII, R = H if guanine interacts with glyoxal and R = CH, or CH,CH (OC,H,) if it reacts with pyruvaldehyde or kethoxal, respectively (174,175).New five-membered rings are also formed in the reactions of guanosine and guanine with ninhydrin (174). 3. With glyoxal (177)and kethoxal (178,l79),the reactions proceed rather rapidly. Thus nonfractionated tRNA reacts with kethoxal a t 37°C and pH 5.0 to give a quantitative modification of guanine residues in about 15 hours with no MgZfand 30 hours in the presence of Mg2+(178). 4. I n principle, l12-dicarbonyl compounds are presumed to affect the secondary structure, just as does formaldehyde, by reacting only with such bases as are localized in single-stranded or unwound double-stranded RNA sites and preventing renaturation in the modified segments (178, 179). However, a very short-lived and limited unwinding, taking place in the fluctuating RNA structure, suffices to cause condensation with 1,2dicarbonyl compounds, which react readily to form a stable product. It appears that even stabilization by M g + ions does not provide for the retention of the secondary structure in the presence of kethoxal (178, 179).Only a t the start of the reaction (30 minutes, pH 7, 37"C, Mg2+) is it possible to locate those tRNAPh' sites that have reacted with [3H]kethoxal without destroying the tRNA conformation (179). 5. l12-Dicarbonyl compounds and formaldehyde are used in different fields. The former are never used for fixation, their mutagenic activity is unknown but kethoxal has been successfully used for labeling and identification of exposed guanine tRNA residues (179,180).On the other hand, the carcinostatic effect and the inactivation of DNA replication are observed under the action of both formaldehyde (or methylene dinucleotides) (2, 43) and l12-dicarbonyl compounds (181,182). Covalent cross-links, hindering the separation of complementary DNA chains on denaturation, are formed on DNA treatment with oxidized spermine [ dialdehyde, OHC (CH,) ,NH (CH,) ,NH (CH,) ,CHO] but are absent when DNA is treated with oxidized spermidinc [monoaldehyde, OHC(CH,),NH (CH,),NH,] (183,184). It follows that both carbonyl

42

M. YA. FELDMAN

groups of oxidized spermine are needed for cross-links. The reaction is nonspecific, for the carbonyl groups of oxidized spermine react with all bases containing amino groups and, though to a considerably smaller extent, with thymine and uracil (186, 184). Another dialdehyde, glutaric aldehyde (OHC (CH2);
B. Difunctional Alkylating Agents4 Formaldehyde is the simplest difunctional reagent. It is of interest to compare the modification of nucleic acids by formaldehyde with the effects produced by other conventional difuctional compounds, such as , aliphatic nitrogen mustard butadiene dioxide (CH,-CH-CH-CH,)

\ /

\/

0 0 HN2 [bis (2-chloroethyl)methylamine, CH,N (CH,CH,Cl) ,] and mustard gas [bis (2-chloroethyl)sulfide, S (CH,CH,Cl) 2]. Reacting with the N-7 of guanine residues in nucleic acids, these compounds give rise to products of monofunctional as well as bifunctional alkylation. Diguanyl derivatives of butadienc dioxide and HN2 are the most chemically studied compounds. The physicochemical and biochemical consequences of bifunctional alkylation of nucleic acids have been most thoroughly investigated in experiments with mustards (188-192). Difunctional alkylation proceeds with intermediate formation of the monoalkyl derivative (170,38, 193). The secondary structure does not Only those rcfercncrs nre given in this srihscction thnt, are not found in the reviews (170, 38).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

43

prevent the action of alkylating agents. Reacting with the native doublestranded DNA, the difunctional agent forms both interchain and intrachain cross-links (192,194). Modification of nucleic acids by difunctional alkylating agents offers the following advantages over modification by formaldehyde. First, reactions with alkylating agents are very fast, less than 1 hour's treatment with mustard gas (37"C, pH 7) giving the necessary effect in many cases. Second, cross-links are specifically formed only between guanine residues [although the poly(A) reaction with mustard gas gives, together with monofunctional alkylation products, an additional product of difunctional alkylation, presumably bis (adenin-l-ylethyl) sulfide (198)1. Third, it is often possible to compare the difunctional alkylating agent and the corresponding monofunctional agent, for instance, mustard gas, bis (2-chloroethyl) sulfide, and hemisulfur mustard, 2-chloroethyl 2-hydroxyethyl sulfide. Compared to formaldehyde, difunctional alkylating compounds display a number of disadvantages, two of which are of particular importance. First, together with diguaninyl derivatives, a great number of stable by-products of monofunctional alkylation are formed. Thus, 20 minutes' treatment of salmon sperm DNA with mustard gas (37", pH 7.2) gives the following alkylation products (190) : bis (P-guanin-7-ylethyl) sulfide, 2270 of total alkylated bases ; 7- (2-hydroxyethylthioethyl) guanine, 59% ; 1- (2-hydroxyethylthioethyl) adenine, 4% ; 3- (2-hydroxyethylthioethyl) adenine, 15%. Second, alkylation reactions are complicated by hydrolytic release of the alkylated purines. Subsequent chain fission of nucleic acid should also not be ignored, however negligible. Comparison of the biological effects of mono- and difunctional alkylating agents in parallel experiments shows what effects are mainly or partly due to the formation of cross-links. The formation of cross-links in DNA results in the inactivation of bacteriophages containing doublestranded DNA (198, 195, 196),exerts a pronounced cytostatic (carcinostatic) effect, and inhibits DNA replication. The effects of difunctional alkylating agents in vivo are observed in both microorganisms (191, 197) and mammalian cells (198).The compounds exert only a weak action on the synthesis of RNA and protein, just as do corresponding monofunctional agents. The part played by each type of cross-linking, intra- and interstrand, is as yet unknown. There is not yet enough evidence to clarify the role of cross-links in mutagenesis induced by alkylating agents. The mechanism of reparation of defects caused by alkylating agents in vivo also requires further study (191, 198). Although many important problems await elucidation, the information available as to the action of difunctional alkylating agents on nucleic

44

M. YA. FELDMAN

acids in vitro and in vivo is very useful for a general biological interpretation of the nucleic acid modifications caused by difunctional agents.

VII. Conclusion Forrnaldehydc is not only one of thc most useful, but also one of the most extensively studied, agents modifying nucleic acids. It is with formaldehydc that the relationship between the modifying agent and the secondary polynucleotide structure appears to have been most thoroughly invcstigatcd. Thc main field of formaldehyde application is fixation. Formaldehyde fixes two opposite structural states, denatured and native. The denaturation of nucleic acid is fixed by the formation of primary products, methylol derivatives. Thc native macrostructure of the polymer is fixed by methylenc cross-links formed in the secondary reaction. For each fixation, suitable conditions must be carefully chosen. Otherwise, mixed effects arc produccd, which lead to incorrect intcrpretations of results. New and important applications of formaldehyde fixation are to be expected, such as fixation of different tRNA conformations and of oncogenic viruses for vaccine preparation (199). The possible biological effects of a new group of analogs of nueleic componcnts, methylene dinucleotides, are also of great interest.

ACKNOWLEDGMENT The author wishes to thank Dr. E. I. Budowsky for fruitful discussion of some sections of the review.

REFERENCES 1. J. E. Salk and J. 13. Gori, Ann. N . Y . Acnd. Sci. 83, 609 (1960). 2. G. Weitrel, F. Schneider, A.-M. Fretzdorff, K. Seynsche and H. Finger, HoppeSeyler’s Z . Physiol. Chem. 334, 1 (1963). 3. I. A. Rapoport, Dokl.Akad. Nnuk SSSR 54, 65 (1946). 4 . W. Ratnayake, Genet. Res. 12, 65 (1968). 6. H. Fraenkel-Conrat, BBA 15, 307 (1954). G. R. Hnselkorn and P. Doty, JBC 236, 2738 (1961). 7. G. Zuibay and R. Marciello, BBRC 11, 79 (1963). 8. L. Grossman, S. S. Levine and W. S. Allison, JMB 3, 47 (1961). 9. J. T. Penniston and P. Doty, Biopolymers 1, 145 (1963). 10. T. Alderson, Mutalion Res. 1, 77 (1964). 11. M. Staehelin, BBA 29, 410 (1958). 19. M. Ya. Feldmnn, Biokhimiyn 25, 563 (1960). 13. T.Alderson, Nature (London) 187, 485 (1960). 14. J. F. Walker, “Formaldehyde,” 3d ed. Reinhold, New York, 1964. 16. M. Ya. Feldmnn, Biokhimiya 27, 378 (1962). 16. M. Yn. Feldmnn, BBA 149, 20 (1967).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

45

17. M. Ya. Feldman, Bwkhimiyn 23, 917 (1958). 18. E. J. Eyring and J. Ofengand, Bchem 6,2500 (1967). 19. N. N. Aylward, J. Chem. SOC.B 11. 627 (1966). 20. S. Lewin, Expeiientiu 20, 666 (1964). 21. S. Lewin, J . Chem. SOC. p. 1462 (1962). 22. S. Lewin, J . Chem. Soc. p. 792 (1964). 23. S. Lcwin and M. A. Barncs, J. Chem. Soc. B 13. 478 (1966). 24. D. E. Hoard, BBA 40, 62 (1960). 26. A. M. Michelson and M. Grunhcrg-Manago, BBA 91, 92 (1964). 26. R. E. Cline, R. Fink and K. Fink. J-4CS 81, 2521 (1959). 27. A. H. Alegria, BBA 149, 317 (1967). 2s. K. H. Scheit, I’etrrrhetlroii Lett. 11. 1031 (1965). 29. C. L. Stevens and A. Rosenfeld, Bchem 5, 2714 (1966). 30. M. Ya. Feldman, Biokhimiyn 29, 720 (1964). 31. D. L. Woodhouse, Nature (Loiitlon) 192, 336 (1961). 3.2. H. Boedtker, B c h m 6, 2718 (1967). 33. German Patent 106493 (1897). 34. B. Skowro6skaSerafinowa, Roczn. Chem. 29, 932 (1955). 36. W. Pfleiderer, F. Sigi and L. Grozinger, Chem. Ber. 99, 3530 (1966). 3G. G. W. Kenner and A. Todd, in “Heterocyclic Compounds” (R. C. Elderfield, ed.), Vol. 6, p. 234. Wiley, New York, 1957. 37. R. K. Robins, in “Heterocyclic Compounds” (R. C. Elderfield, ed.), Vol. 8, p. 162. Wiley, New York, 1967. 3s. N. K. Kochetkov, E. I. Budowsky, E. D. Sverdlov, N. A. Simukova, M. F. Turchinsky and V. N. Shibaev, “Organichcskayn khimiya nucleinovikh kislot.” Khimiya, Moskva, 1970. 89. German Patent 667542 (1938). 40. German Patent 669188 (1938). 41. US. Patent 2211710 (1940). 4.2. M. Ya. Feldmnn, unpublished data. 43. M. Ya. Fcldmnn, E. S. Zalmanson and L. N. Mikhailova, Mol. Biol. 5, 847 (1971). 44. C. D. Jardetzky and 0. Jardetzky, JACS 82, 222 (1960). 46. H. A. Szimansky and R. E. Yelin, “ N M R Band Handbook.” IFI/Plenum, New York, 1968. 4G. W. E. Cohn, in “The Nucleic Acids” (E. Chargaff and J. N. Davidson, cds.), Vol. 1, p. 211. Academic Press, New York, 1955. 47. M. Ya. Feldman, unpublished results. 4s. M. Yn. Fcldman and M. R. Shubinn. Dokl. Akud. Nnuk SSSR 174, 236 (1967). 49. R. F. Beers, Jr. and R. F. Steincr, Nature (London) 179, 1076 (1957). 60. G. D. Fasman, C. Lindblow and L. Grossman, Bchem 3, 1015 (1964). 61. German Patent 139907 (1901). 62. H. Fraenkel-Conrat, JACS 78, 882 (1956). 63. A. Gierer and G. Schramm, Nature (London) 177, 702 (1956). 64. M. YR. Feldman, Biokhimiyn 30, 203 (1966). 66. M. Ya. Feldman, Biokhimiyn 25, 937 (1960). 6G. V. D. Axelrod, M. Ya. Feldman, I. I. Chuguev and A. A. Baycv, BBA 186, 33 (1969). 67. B. D. Hall and P. Doty, JMB 1, 111 (1959). 6s. S. Zamenhof. H. E. Alexnnder and G. Leidy, J. Exp. Med. 98, 373 (1953).

46 69. C. GO. R. 61. C.

M. YA. FELDMAN

J. Collins and W. R. Guild, BBA 157, 107 (1968).

L.Sinsheimer, J M B 1, 43 (1959). A. Thomas, Jr. and K. I. Berns, J M B 4, 309 (1962).

62. P. Sheldrick and W. Szybalski, J M B 29, 217 (1967). 63. D.Freifelder and P. F. Davison, Biophys. J. 3, 49 (1963). G4. A. J. van tler Eh, I,. M '. van Kesteren nnd E. F. J. van Rruggm. BBA 182, 530 (1969). 06. H. Utiyama and P. Doty, Bchem 10, 1254 (1971). 66. A. Rosenfeld, C. L. Stevens and M. P. Printz, Bchem 9, 4971 (1970). 67. G.Zubay and R. Marciello. BBRC 11, 79 (1963). 6s. G. D. Fasman, C. Lindblow and E. Seaman, J M B 12, 630 (1965). 69. D. B. Millar and M. MacKrnzir, Bcliem 6, 2520 (1967). 70. R. A. Cox, K. Kanagalingam iind E. S. Sutherland, BJ 120, 549 (1970). T i . M. Edelman, I. M. Verma, D. Saya and U. Z. Littauer, BBRC 42, 208 (1971). 72. H.Boedtker, BBA 240, 448 (1971). 73. V. S. Mayo and S. R. de Kloet, BBA 247, 74 (1971). 74. P. L. Whitney and C. Tanford, JBC 237, 1735 (1962). 76. W. M.Stanley, Jr. and R. M. Bock, Bchem 4, 1302 (1965). 76. M.L.Fenwick, BJ 107, 851 (1968). 77. H.Boedtker, J M B 35, 61 (1968). 78. H. Slegers and W. Fiers, FEBS Lett. 7, 55 (1970). 79. H. Slegers and W. Fiers, Biopolymers 9, 1373 (1970). 80. N.K. Sarkar and A. L. Dounce, BBA 49, 160 (1961). Si. K.I. Berns and C. A. Thomas, Jr., J M B 3, 289 (1961). S9. D. Stollar and L. Grossman, J M B 4, 31 (1962). 83. M. Beer and C. A. Thomas, Jr., J M B 3, 699 (1961). 84. R. B. Inman, JMB 18, 464 (1%). S5. V. V. Simonov, N. I. Ryabchenko and A. M. Povcrenny, Mol. Biol. 1, 297 (1967). 86. E. A. C. Follet and L. V. Crawford, J M B 34, 565 (1968). 87. P. H. von Hippel and K.-Y. Wong, J M B 61, 587 (1971). 88. R.J. Cohen and D. M. Crothers, JMB 61, 525 (1971). 89. E. N. Trifonov, N. N. Shafranovskaya, M. D. Frank-Kamenetskii and Yu. S. Lazurkin, Mol. B i d . 2, 887 (1968). 90. Yu. S. Lazurkin, M. D. Frank-Knmenetskii and E. N. Trifonov, Biopolymers 9, 1253 (1970). 91. R. B. Inman and M. Sch~os,J M B 49, 93 (1970). 92. J. T.Penniston and P. Doty, Biopolymers 1, 209 (1963). 93. J. T. Penniston. M. Steward and M. D. Tucker, BBRC 15, 358 (1964). 94. M. K. Iiuklinnora, I,. I,. Kisrlcv and I,. Tu. Frolova, Biokliimiya 28, 1053 (1963). 96. A. S. Girshovich, M. A. Grachev, N. I. Komnrova and N. I. Mensorova, FEBS Lett. 14, 195 (1971). DG. L. J. Saidel, S. Leitzes and W. H. Elfring, Jr., BBRC 15, 409 (1964). 97. H. F. Lodish, J M B 50, 689 (1970). 98. J. C.Pinder and W. B. Gmtzer, Bchem 9, 4519 (1970). 99. M.Ya. Feldman, Vopr. &fed. Khim. 9, 232 (1963). 100. D.Brutlag, C. Schlehuber and J. Bonner, Bchem 8, 3214 (1969). 101. M.Roberts and I. 0. Walker, BBA 199, 184 (1970). iO2. M.Levy and D. Silberman, JBC 118, 723 (1937).

REACTIONS O F FORMALDEHYDE WITH NUCLEIC ACIDS

47

103. E. H. Frieden, M. S. Dunn and C. D. Coryell, J. Phys. Chem. 47, 10 (1943). 104. D. French and J. T. Edsall, Advan. Protein Chem. 2, 277 (1945). 105. B. P. Zhantalai and Ya. I. Tur’yan, Kinet. Kntal. 3, 325 (1962). 106. Ch. J. Martin and M. A. Marini, JBC 242, 5736 (1967). 107. H. Nitschmann and H. Hndorn, Helv. Chim. Acta 27, 299 (1944). 10s. H. Fraenkel-Conrat and D. Mcrham, JBC 177, 477 (1949). 109. Ii. I. Stracliitskii, iir “Voprosi medicinkoi lihimii” (8. R . Mardnslicv. cd.), Vol. 1, p. 292. Mcdgiz, Moskva, 1949. 110. M. P. Drake and A. Veis, Bchem 3, 135 (1964). 111. D. W. Bannister, BJ 114, 509 (1969). 112. P. V. Hauschka and W. F. Harrington, Bchem 9, 3734 (1970). 113. H. Nitschmnnn and H. Laucncr, Helv. Chim. A d a 29, 184 (1946). 114. H. Fraenkel-Conrat and H. Olcott, JACS 70, 2673 (1948). 115. H. Fraenkel-Conrat and H. Olcott. JBC 174, 827 (1948). llG. J. Blass, BuI1. SOC.Chim. p. 3120 (1966). 117. R. D. Haworth, D . Peacock, W. Smith and R. MacGillivry, J. Chem. SOC. p. 2972 (1952). 11s. J. Blass, B. Bizzini and M. Raynaud, C . R . Acad. Sci. 261, 1448 (1965). 119. J. Blass, B. Bizzini and M. Raynaud, Bull. SOC.Chim. p. 3957 (1967). 120. K. H. Gustavson, Advan. Protein Chem. 5, 353 (1949). 121. N. I. Egorkin, M. A. Mamedov and 0. D. Rokhwarger, “Formaldegidnoye dubleniye.” Gizlegprom, Moskva, 1957. 12.2. J. Blass, B. Bizzini and M. Raynaud, Ann. Znst. Pasteur IlG, 501 (1969). 123. M. L. Petterman and A. M. Pappenheimer, Jr., J. Phys. Chem. 45, 1 (1941). 124. J. F. Largier and F. J. Joubcrt, BBA 20, 407 (1956). 125. J. Blass, Biol. Me‘d. 53, 202 (1964). 126. H. Fraenkel-Conrat, in “Amino Acids and Proteins” (D. M. Greenberg, ed.), p. 532. Thomas, Springfield, Illinois, 1951. 127. P. Desnuclle, in “The Proteins. Chemistry, Biological Activity and Methods” (H. Neurath and K. Bailey, eds.), Vol. 1, Part A, p. 87. Academic Press, New York, 1953. 1%. F. W .Putnam, in “Thc Proteins. Chemistry, Biological Activity and Methods” (H. Neurath and K. Bailey, cds.), Vol. 1, Part B, p. 893. Academic Press, New York, 1953. 129. R. A. Cox, BJ 114, 743 (1969). 130. M.Tal, BBA 224, 470 (1970). 131. M. Tal, Bchem 8, 424 (1969). 132. A. S. Spirin, N. V. Belitsina and M. I. Lerman, J M B 14, 611 (1965). 133. H . E. Huxley and G. Zubay, JMB 2, 10 (1960). 134. S. T . Bayley, JMB 8, 231 (1964). 135. Yu. A. Rornakov and G. V. Bozhko, Biokhimiya 32, 471 (1967). 1SG. M. E. Lamm, L. Childers and M. K. Wolf, J. Cell Bio2. 27, 313 (1965). 137. N. Feder and M. K. Wolf, J . Cell Biol. 27, 327 (1965). 13S. A. V. Furano, D. F. Bradley and L. G. Childers, Bchem 5, 3044 (1966). 139. Yu. N. Kosaganov, M. I. Zarudnaya, Yu. S. Lazurkin, M. D. Frank-Kamenetskii, R. Sh. Beabenlashvili and L. P. Savochkina, Nature (London) New Biol. 231, 212 (1971).

140. W. Doerfler and A. K. Kleinschrnidt, JMB 50, 579 (1970). 141. A. S. Bayev, Yu. L. Lyubchenko, Yu. S. Lazurkin, E. N. Trifonov and M. D. Frank-Kamenetskii, Mo2. Biol. 6, 760 (1972).

48

M. YA. FELDMAN

142. S. R. de H o e t , V. 8. Mayo and B. A. G . Andretin. BBRC 40, 454 (1970). 143. J. M. Bishop and G. Koch, JBC 242, 1736 (1967). 1 4 . A. M. Poverenny, T. L. Aleinikova and A. S. Saenko, Biokhimiya 31, 1150 (1966). 146. R. L. Sinsheimer, JMB 1, 37 (1959). 146. W. Sauerbier, Nature (Loiidon) 188, 327 (1960). 147. T. I. Tikhonenko and E. N. Dobrov, JMB 42, 119 (1969). 148. World Health Organization, “Human Viral and Rickettsia1 Vaccines,” Technical Report Series, No. 325. Geneva, 1966. 149. Ch. Andrewes, “Viruses of Vertebrates.” Bailliere, Tindall and Cox, London,

1964.

160. Chemical Inactivation of Viruses, Ann. N . Y . Acad. Sci. 83, No. 4, Part 11, 564653 (1960). 161. E. Maron, C. Shiozawa, R. Arnon and M. Scla, Bchem 10, 763 (1971). 162. F. A. Anderer, M. A. Koch and S. D. Hirschle, Eur. J . Immunol. 1, 81 (1971). 163. R. Markham, R. E. F. Matthews and K. M. Smith, Nnture (Londoti) 162, 88 (1948). 164. M. Staehelin, BBA 31, 448 (1959). 166. A. Ross and W. Stanley, J . Gen. Physiol. 22, 165 (1938). 166. R. Haas, R. Thomssen, V. Dostal and E. Ruschmann, Arch. Ges. Virusforsch. 9, 470 (1959). 167. T. Alderson, Nature (London) 215, 1281 (1967). 168. G. D. Zasukhina and I. A. Rapoport, Genetika 5, 33 (1965). 169. Yu. Z. Gendon, “Genetika virusov cheloveka i zhivotnikh.” Nauka, Moskva, 1967. 160. E. Englesberg, J. Bactenol. 63, 1 (1952). 161. F. H. Dickey, G. H. Cleland and C. Lotz, PNAS 35, 581 (1949). 162. F. H. Sobels, Radial. Res. SuppZ. 3, 171 (1963). 163. I. H. Herskowitz, Science 112, 302 (1950). 164. C. Auerbach and H. Moser, 2. Vererbungslehre 85, 479 (1953). 166. H. Nafei and C. Auerbach, 2. Vererbungdehre 95, 351 (1964). 16G. T. Alderson, Nature (London) 191, 251 (1961). lG7. A. F. E. Khishin, Mutation Res. 1, 202 (1964). 168. W. E. Ratnayake, Mutation Res. 9, 71 (1970). 169. W. D. Kaplan, Science 108, 43 (1948). 170. P. D. Lawley, This Series 5, 89 (1966). 171. R. Shapiro, This Series 8, 73 (1968). 172. A. Loveless, “Genetic and Allied Effects of Alkylating Agents.’’ Butterworths, London, 1966. 173. R. K. Lyakyavichus, Uspekhi Sour. Genet. 1, 165 (1967). 174. R. Shapiro and J. Hnchmann, Bchem 5,2799 (1966). 176. R. Shnpiro, B. I. Cohen, 8.5.Shiuey and H. Maurer, Bchem 8, 238 (1969). 176. R. Shapiro, B. I. Cohen and D. C. Clagett, JBC 245, 2633 (1970). 177. N. E. Broude, E. I. Budowsky and N. K. Kochetkov, MoZ. BWZ. 1, 214 (1967). 178. M. Litt and V. Hancock, Bchem 6, 1848 (1967). 179. M. Litt, Bchem 8, 3249 (1969). 180. M. Litt, Bchem 10, 2223 (1971). 181. F. A. French and B. L. Freedlander, Cancer Res. 18, 172 (1958). 18.3. 0. L. Klamerth, BBA 155, 271 (1968). 183. U. Bachrach and G. Eilon, BBA 145, 418 (1967).

REACTIONS OF FORMALDEHYDE WITH NUCLEIC ACIDS

49

l S 4 . U. Baclirarh and G. Eilon, BBA 179, 473 (1969). 186. G.Eilon and U. Buclirdi, BBA 179, 464 (1969). 186'. U.Bachrach and S. Pcrsky, BBA 179, 484 (1969). l S 7 . B. W. Kimes and D. It. Morris, BBA 228, 235 (1971). 188. K . W. Kohn, C. I,. Spears cind P. Dots, JMB 19, 266 (1966). 1259. K. W.Kohn and D. M. Green, J M B 19, 289 (1966). 190. P. D.Lawley and P. Brookcs, J M B 25, 143 (1967). 191. P. D.Lawley and P. Brookrs, BJ 109, 433 (1968). 192. P. D. Ltnvley, J. H. Lethbridgc, P. A. Edwards iind K.V. Shootcxr, J M B 39, 181 (1969). 193. R . J. Rutman, E. H. I,. Chun and J. Jones, BBA 174, 6f33 (1969). 104. W.G . Flnmm, N. J. Brrnlieim and I,. Fishbein, BBA 224, 657 (1970). 196. W.G. Verly and I,. Brnkirr, BBA 174, 674 (1969). 1911. L. Brakier and W-.G. Vrrly, BBA 213, 296 (1970). 197. S. Venitt. P. Brookes and P. D. La\vlcy, BBA 155, 521 (1968). 198. B. D.Reid and I. G.Walker, BBA 179, 179 (1969). 199. K.Takizawit and T. Yaniamoto, Gatin 61, 605 (1970).