Structural investigations on dehydrogenases by means of difference spectrophotometry

BIOCHIMICA ET BIOPHYSICA ACTA BBA

65300

STRUCTURAL INVESTIGATIONS ON DEHYDROGENASES BY MEANS OF DIFFERENCE

SPECTROPHOT~METRY

S. LIBOR, P. ELODI AND Z. NAGY Institute of Biochemistry and Laboratory for Research of Chemical Structure, Hungarian Academy of Scienoesc Budapest [Hungary} (Received April z znd, 1965)

SUMMARY I. The distribution and localization of tyrosine in glyceraldehyde-j-phosphate dehydrogenase (n-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) and in lactate dehydrogenase (r-lactate.Na.Df oxidoreductase, EC 1.1.1.27) were studied by difference spectrophotometry through spectrophotometric titration, solvent perturbation and iodination. 2. In both dehydrogenases the alkaline dissociation of some phenolic hydroxyl groups is anomalous. The apparent pK's are 11.3 and II.S, and the titrations are irreversible. 3. As measured by the solvent perturbation method, maxima of 45 and 50% of the tyrosyl residues may be located on the surfaces of the glyceraldehyde-g-phosphate dehydrogenase and lactate dehydrogenases respectively. 4. About 65 and 80% of the tyrosyl groups are accessible to iodine in glycine buffer (pH 9.2). The remainder becomes available to iodine only after the proteins have been denatured with concentrated urea. 5. On the basis of the above findings the structural composition of these dehydrogenases is discussed.

INTRODUCTION

In various investigations on glyceraldehyde-g-phosphate dehydrogenase (nglyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) and on lactate dehydrogenase (t-lactate.Na.Dt oxidoreductase, EC 1.1.1.27) it was found that their relative instability to some changes in the environment might be attributed to the lack of strong stabilizing forces (e.g. disulfide bridges) in their structure. In previous papers>" on the basis of the effect of non-polar solvent and detergent, it was emphasized that the hydrophobic forces could have an important role in the maintenance of the enzymatically active structures of these proteins. This idea is also supported by the fact that both glyceraldehyde-j-phosphate dehydrogenase Biochim, Biophys. Acta,

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DIFFERENCE SPECTROPHOTOMETRY ON DEHYDROGENASES

and lactate dehydrogenase are composed of large amounts of non-polar amino acid side-chains-.", A study of the aromatic chromophores, such as tyrosine, can supply information about the relationship between the polar and non-polar parts of the protein molecule because these amino acids may be located in both regions. Moreover, their alterations caused by changes in the environment can be followed spectrophotometrically. The purpose of the present investigation was to obtain information about the situation and distribution of tyrosyl residues by comparing the data of spectrophotometric titration, solvent perturbation and iodination of the two dehydrogenases. MATERIALS AND METHODS

Glyceraldehyde-j-phosphate dehydrogenase was isolated and re-crystallized four times as described earlier", The enzyme was dialyzed against 0.1 M glycine buffer (pH 8.5). Lactate dehydrogenase was isolated according to the method described by JtCSAI 7. Preparations were re-crystallized three times, then dialyzed against 0.05 M phosphate buffer (pH 7.5). The protein contents of the dialyzed samples were determined spectrophotometrically in 0.1 M NaOH by measuring the absorbance at 280 mp, and calculated on the basis of an E~~m at 280 mp of 1.00 and 1.29 for glyceraldehyde-j-phosphate dehydrogenase and lactate dehydrogenase respectively. Protein solutions were kept at

4°· Chemicals Urea was re-crystallized twice from a 70% (vjv) ethanol-water mixture. The concentrated urea solutions were prepared freshly to avoid isomerization. Sodium dodecyl sulfate was purchased from the DuPont-Nemours Co. and recrystallized from ethanol. Ethylene glycol was a Reanal (Budapest) preparation; it was re-distilled under reduced pressure. All other reagents used in this investigation were of reagent grade. Solutions were prepared with glass-distilled water.

Spectrophotometric titration Protein solutions containing 0.7 or 1.4 mg protein per rnl were preincubated for I h at room temperature in 0.1 M glycine-NaOH buffer or in 0.1 M glycine-HCl buffer of different pH's for the determination of the change in the absorbance as a function of pH. Readings were taken in r-em silica cells in a Spectromom 201 spectrophotometer. After the spectrophotometer readings had been taken, the pH of each solution was measured by using a Radelkisz "Blood pH meter" with an accuracy of ±0.05 pH unit.

Difference spectra Difference spectra between 260 and 3IO mp were measured with a Unicam SP 700 recording spectrophotometer. In the blank cell glyceraldehyde-j-phosphate deBiochim, Biophys. Acta,

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hydrogenase and lactate dehydrogenase solutions in 0 .1 M glycine buffer (pH 8.5) and 0.05 M phosphate buffer (pH 7.5) were used respectively, and regarded as "neutral" references throughout the present paper.

Solvent perturbation difference spectra These were measured by HERSKOVlTS AND methods in the presence of 20% ethylene glyc ol.

LASKOWSKI'S

tandem cuvette

Iodination Iodination was carried out with KIa under optimum conditions'' for the iodinat ion of tyrosyl residues in proteins between pH 9.2 and 9.5 at room temperature. The KIa stock solution was prepared by dissolving iodine in a 0.15 N KI solution up to 0.1 N final concentration of iodine. The concentration of these solutions was determined by thiosulfate titration. The excess of iodine in the iodination experiments was removed by the addition of 0 .1 N arsenite solution. All calculations are based on a molecular weight of 140 000 for both dehydrogenases. This value was found earlier for glyceraldehyde-j-phosphate dehydrogenase-v and it is assumed for swine-muscle lactate dehydrogenase on the basis of the molecular weight of lactate dehydrogenases of other origin». Amino acid solutions To compare the chromophoric properties of "free" tyrosine with those oftyrosyl groups in proteins a tyrosine-tryptophan amino acid mixture was made in the molar ratio found in glyceraldehyde-j-phosphate dehydrogenase, namely 36 moles of tyrosine and 16 moles of tryptophan per mole of glyceraldebyde-j-phosphate dehydrogenase.

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F ig. I. pH-difference spectra of drogenase bet w een 2 2 0 and 2 8 0 Neutral samples (see METHODS) deh ydrogenase ; - - - . lactate

gtyceraldehyde-j-phosphate dehydrogenase and lactate deh ymfl.. A, in 0 .1 N NaOH; B, in o.z N glycine buffer (pH 2.0).

w ere taken as references. - - , glyceraldehyde-g-phosphate dehydrogenase.

DIFFERENCE SPECTROPHOTOMETRY ON DEHYDROGENASES RESULTS

The difference spectra of the acidic and alkaline samples, against the "neutral" reference solutions, from 220 to 280 mfl are shown in Fig. I. Alkaline difference spectra The alkaline difference spectra (Fig. IA) exhibit a large maximum at about 243 mfl and a minimum at 228 tau: The maximum at this wavelength is due to a combined effect of different interactions (ionization of tyrosine and sulfhydryl groups and possibly of other side-chains, changes in peptide hydrogen bonding, etc.) . The difference spectra of the two dehydrogenases in 0 .1 M NaOH between 260 and jro mp are shown in Fig. zd. Both dehydrogenases exhibit a maximum around 296 mfl and a shoulder at 291 mp. The difference observed at these wavelengths is attributed to the phenolic dissociation of tyrosyl side-chains. The number of tyrosine residues in both dehydrogenases can be calculated from the molar extinction coefficients" of ionized phenolic groups at 297 rop,: .daM = 2.33 . 103. The molar extinction differences and the number of tyrosines calculated are shown in Table 1. This tabel shows that, if the number of tyrosines is calculated from the difference between the native "neutral" sample and the alkaline sample, the tyrosine content is underestimated: about two-thirds of the tyrosines of glyceraldehyde-gTABLE I THE MOLAR EXTINCTION DIFFERENCES AT THE TYROSINE CONTRNT

Enzyme

297 mil

OF ALKALINE DEHYDROGENASE SOLUTIONS AND

Measured against "native" reference

Measured against "denatul'ed "reference

.deA: at 297 m,u Number of

tle,'l/ at 297 mflo Number of

tyrosil,es calculated Glyceraldehyde-j-phosphate dehydrogenase 58 Lactate dehydrogenase 49

000 000

±2 ±2

500 25 600 2 I

± ±

1. I 1.2

Number of tyrosines from amino acid analysis

tyrosines calculated

83

000 67°00

± 4 500

± 4000

36 ± 1.9 28 ± 1.7

36 (ref. II) 30 (ref. 12)

phosphate dehydrogenase and about three-fourths of the tyrosines oflactate dehydrogenase are found. Therefore we assume that the neutral protein solutions are not proper references for the determination of the total number of tyrosines in these dehydrogenases. However, when, instead of the neutral solutions, urea- or aciddenatured protein solutions are used as references, the difference at 297 mfl corresponds to the number of tyrosine residues found by amino acid analysisv" with a fairly good agreement. This can be attributed to the fact that in native proteins there is a red shift due to the modifying effect of the structure on the chrornophores. "Denatured" difference spectra Difference spectra were measured under the following denaturing conditions: in the presence of 6 M urea, or with sodium dodecyl sulfate (r:r, w/w) in neutral buffers, and at pH 2 in 0.1 M glycine buffer. The results are shown in Figs. I and 2. Biocbim, Biophys. Acta.

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The acidic difference spectra at the shorter wavelengths (Fig. IE) show, in comparison with the alkaline ones, a trough with a minimum at about 232 mft. The origin of the difference minimum at 232 mft has not yet been completely clarified; GLAZER AND SMITH12,13 assume that it is due primarily to changes in peptide-chain conformation. The difference at the longer wavelengths obtained by using different denaturing agents show a close relationship, as can be seen in Fig . 2 , curves a-c. Apparently similar difference spectra can be obtained when the protein is subjected to low pH,

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Fig. 2 . Difference spectra of glyceralclehydc-3-phosphate dehydrogenase and lactate dehydro, genase between 260 and 310 mp.. A, gtyceraldehyde-g-phosphatc dehydrogenase; B, lactate dehy drogenase: a, in the presence of sodium dodecyl sulfate (I : I, W /w); b, in o. I M glycine buffer (pH 2.0) ; c, in 6 M urea (pH 8.5); d, in o. IN NaOH. Curves a-d were taken against neutral references; e, in 6 M urea in the presence of 0.1 N NaOH, against a neutral reference in 6 M urea.

detergent, and concentrated urea. Although these agents act in different ways on the proteins, there is a common feature in their action, namely all ofthem eventually destroy the organized protein fabric. The decrease in absorbance between 270 and 30 0 mft is generally referred to as a "denaturation blue shift". In the denatured state those chromophoric groups that are partly or completely buried in the interior of the native protein come into contact with the surrounding solvent U - 10 . At acidic pH there may be a small contribution caused by the electrostatic charge effect on aromatic chromophores, but the extent of this contribution in the case of proteins is usually so small that it is practically negligible 16,17 . Spectrophotometric titration

The absorbance differences as a function of pH between 2 and I3 are shown in Fig. 3. The pH of the solutions was adjusted with O.I M glycine buffers of different pH's. The titration was carried out from the neutral pH in both the acidic and alkaline B iochim, Biopbys. Acta.

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DIFFERENCE SPECTROPHOTOMETRY ON DEHYDROGENASES

A

B

2

12

2

4

6

8

10

12

pH Fig. 3. Spectrophotometric titration of glyceraldehydc-j-phosphate dehydrogenase and lactate dehydrogenase between pH 2 and 13 at 292 m~. A, glyceraldehyde-j-phosphate dehydrogenase; B, lactate dehydrogenase. Open signs represent the forward titration and closed signs the reverse titration. Circles, without urea; triangles, in the presence of 6 M urea.

direction (forward titration), and also from pH 2 and 13 towards neutral (reverse titration) . The alkaline dissociation of phenolic hydroxyl groups of both dehydrogenases is anomalous. The dissociation begins at about pH 9.S-ro.o and the apparent pK values of the dissociation are quite high, II,3 and II.5 for lactate dehydrogenase and glyceraldehyde-j-phosphate dehydrogenase respectively. The dissociation is not reversible, and the titration curves obtained in the forward direction differ from those of the reverse direction whether we start from pH 13 or from pH 2. In the reverse titrations, when the pH approaches 5 from the acidic side or 9.6 from the alkaline side, both dehydrogenases precipitate. In the reverse titrations from both directions the same extinction differences are obtained at a given pH independently of the original pH of the solution and from the direction of the change in pH. This observation suggests that the effect of high pH on the structure of these dehydrogenases is related to that of the low-pH effect in this respect. The lack of reversibility and the anomalous high pK of ionization indicate the presence oftyrosyl groups buried in the interior of the polypeptide chains. When the alkaline titration is carried out in the presence of 6 M urea the anomalous character of the curves disappears. The pK values decrease to about IO.3-IO.5, characteristic of tyrosine in polypeptide chains ionizing normally-", and the titrations are reversible.

Solvent p'erturbation spectra According to HERSKOVITS AND LASKOWSKI 8 , polyalcohols such as ethylene glycol, glycerol, sucrose, etc. cause a change in the absorption of aromatic chromoph ores between 270 and 290 mfl without influencing the conformation of polypeptide chains. By means of the tandem-cell methods, the perturbation spectra of the deBioobim. Biophys, Acta,

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49°

S. LIBOR , P. ELODl, Z. NAGY

hydrogenases were measured in the presence of 20% (vjv) ethylene glycol. For the determination of "free" and " bu ried" tyrosyl residues the measurements were carried out in neutral buffers, without and with 6 M urea, and in O.I M glycine buffer (pH 2.0). The experiments wer e also carried out with an amino acid mixture. The results are presented in Fig. 4. The extent of perturbation with glyceraldehyde-g-phosphate dehydrogenase in "neu t ral" buffer (Fig. ¥r) amounts to about 45 % of the value obtained in the presence of urea or at acidic pH (Figs. 4A2 and 4A3). The perturbation spectrum of the amino acid mixture (Figs. 4(1 and 4(3). howe ver , coincides fairly well with those of denatured glyceraldehyde-j-phosphate dehydrogenase. In the case oflactate dehydrogenase (Fig. 4£) about 55 % ofthe tyrosines are in contact with ethylene glycol without a previous urea or acid treatment.

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Fig. 4. Solvent perturbation spectra. of glyceraldehyde-g-phosphate dehydrogenase, lactate dehydrogenase and an amino acid m ixture (see METHODS) in the presence of 20% ethylene glycol. A, glyceraldehyde-3-phosphate dehydrogenase ; B , lactate dehydrogenase ; C, amino acid mixture. - - , (I) , in 0.1 M glycine buffer (pH 8.5); - - - -, (2). in O,I M glycine buffer (pH 2.0 ) ; ' - ' - ' , (3), in 6 M urea (pH 2.0 ) .

The values obtained for tyrosines accessible to ethylene glycol in neutral solution represent the upper limit of the reaction of these groups, which might be an overestimation of the number of tyrosines located on the surface of these proteins". It seems to be reasonable that some of the chromophores situated near the surface could also be included in the above values.

Iodination of tyrosines For the differentiation between "accessible" and "inaccessible" tyrosyl groups, iodination appears to be a suitable method. HUGHES AND STREASSLE 9 have reported that between pH 9.2 and 9.5 the tyrosyl groups of human serum albumin ar e specifically iodinated when a slight excess of KIa is applied 23 •24 • The protein :iodine molar ratios were I :130 and I .r ro, for glyceraldehyde-g-phosphate dehydrogenase and lactate dehydrogenase respectively, in the present investigation. The iodination was carried out in 0 .1 M glycine buffer (pH 9.2) at room temperature. The iodination of tyrosyl groups can be followed by measuring the increase in Biochim, Biophys. A eta,

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DIFFERENCE SPECTROPHOTOMETRY ON DEHYDROGENASES

A

B

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O.B 0.6 0.4

0.2

0 260

300

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Fig. 5. Difference spectra of thc iodinated glyceraldehyde-j-phosphate dehydrogenase and lactate dehydrogenase. A. glyceraldehyde-g-phosphate dehydrogenase; B, lactate dehydrogenase. Protein concentration 7oo/lg/mt. X - - - - - X, in 0.1 M glycine buffer (pH 9.2); .-e, in 6 M urea (pH 9.2). Samples without iodine added were taken as references.

absorbance at 312 mfl, the absorbance maximum of diiodotyrosine. The difference spectra of the iodinated dehydrogenases, in neutral buffers and after denaturation with 6 M urea, are presented in Fig. 5. The curves show that, when iodination was carried out without previous urea denaturation, the absorption difference at the maximum wavelength is about 65 and 80% of that of the denatured samples for glyceraldehyde-j-phosphate dehydrogenase and lactate dehydrogenase respectively. This finding suggests that a part of the tyrosyl residues is not available even for iodine when it attacks the proteins in the native state. When iodination was carried out with TABLE II MOLAR EXTINCTION DIFFERENCES AT 312

mp,

OF IODINATED DEHYDROGENASES AND THE NUMBER

OF DIIODOTYROSINES FORMED PER MOLE OF PROTEIN

Enzyme

In glycine buffer (PH 9.IJ)

In 6 M urea (pH 9.13)

JeM at 3IIJ mp Moles diio-

Jell-! at 3I2 mp

dotyrosine Glyceraldehyde-j-phosphate dehydrogenase Lactate dehydrogenase

160 000 170000

± ±

7000 23.9 4000 24.5

± ±

LI

240000

0.6

200000

±

Moles diiodotyrosine

6000 35.4

± 8000

30.1

±

0·9

± 1.2

the amino acid mixture, no measurable difference was found in the number of diiodotyrosines formed in the presence or absence of urea. For the molar extinction coefficient at 312 mfl of diiodotyrosines a value of 6670 was calculated for both dehydrogenases. This value is higher than that found for other proteins-" ,26. The molar extinction differences measured, and the number of diiodotyrosines calculated therefrom in the presence and absence of 6 M urea, are shown in Table II. The specificity of iodination for the dehydrogenases is not complete because of Biochim, Bioplws. Acta,

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S. LIBOR, P. ELODI, Z. NAGY

the presence of free sulfhydryl groups also reacting with iodine. In the calculation of the proper protein :iodine ratio the number of sulfhydryl groups was also taken into account. The rate of formation of diiodotyrosine was followed in time on the basis of the change in absorbance at 312 mp: A significant difference was found in the velocity of diiodotyrosine formation when iodination was performed in the presence and in the absence of concentrated urea (Fig. 6). The experiment was carried out in the following way. Samples containing 700 flg protein per ml were incubated with KIa in a molar ratio of I :130 and I :IIO at room temperature in 0.1 M glycine buffer (pH 9.2) and in 6 M urea at the same pH. The final volume of the reaction mixtures was 4.9 ml. The iodination was stopped at different time intervals by the addition of 0.1 ml of 0.1 N arsenite solution, and the samples were thoroughly mixed. Then readings were taken at 312 m/-, in r-em cells. The duration of iodination varied from IO sec to I h.

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Fig. 6. Time dependence of the reaction of iodine with dehydrogenases, A, glyceraldehyde-gphosphate dehydrogenase; E, lactate dehydrogenase. 70 0 !Jog protein per rnl. 0 - - -0, in o.r M glycine (pH 9.2); .-e, in 6 M urea (pH 9.2). .

Fig. 6 shows that the extent of iodination of the native samples differs considerably in the first 10-30 sec. After 10 sec of incubation with iodine in the glyceraldehyde-j-phosphare dehydrogenase about 30% of the tyrosines are iodinated whereas for lactate dehydrogenase this value is around 50%, the maximum difference measured in urea being taken as 100%. This difference between the native proteins decreases gradually, and the absorption becomes practically steady after IO min of incubation. At this equilibrium about 65 and 80% of the total number of tyrosines are in the diiodo form in glyceraldehyde-g-phosphate dehydrogenase and lactate dehydrogenase respectively (Table II). The differences in the rate of iodination found between glyceraldehyde-g-phosphate dehydrogenase and lactate dehydrogenase might be ascribed to the difference in the structure of the two dehydrogenases. The iodination of amino acid mixture takes place instantaneously both in the presence and absence of urea. The time dependence of the reaction of iodine with native proteins shows that less tyrosines react instantaneously than were found at the end-point. This might suggest that in the native proteins a part of the accessible tyrosines is able to react only after some Biochim. Biophys. Acta.

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493

previous alteration in the steric structure. The residual 35 and 20 % of tyrosyl residues react only when complete disorganization of the native protein fabric has taken place. DISCUSSION

In the compact, organized, native protein molecule the amino acid side-chains may be situated in a number of different ways. Either they project towards the surrounding solvent medium, or they are folded into the internal parts of the macromolecule , surrounded by the other side-chains referred to as internal environment. An intermediate situation, however, may also exist, in which the side-chains are partly embedded in the interior, not far from the surface. Difference spectrophotometric investigation of the aromatic chromophores can provide information about the location of these side-chains if their distribution is assumed to be statistical. From the ionization of tyrosyl residues, i.e. whether it occurs normally or anomalously, the relative positions of these residues can be determined15 ,16. Generally, when tyrosines dissociate anomalously and their titration is irreversible, it means that these groups are taking part in some interactions or they are buried in the interior of the protein molecule. When so buried, phenolic groups become available to protonation only when a previous destruction of the organized structure of the polypeptide chains has occurred . In the present investigation it was found by spectrophotometric titration that the tyrosyl groups of both dehydrogenases ionize anomalously with high apparent pK's of about II .3 and II.5 as compared with the pK values of 9 .9-10.2 for free tyrosines 2 0 , 21 and about IO.4 for tyrosines in unfolded polypeptide chains-", Further, the titrations are irreversible. These findings suggest that both dehydrogenases contain tyrosines located in the inner part of the molecule. The forward titration curves are S-shaped, and the apparent pK values observed are in fact only the midpoints of the titration curves. From these "mixed" pK values the number of "free" and " bound" t yr osines cannot be calculated, which suggests a gradual instead of stepwise'" ionization of tyrosines having different positions in the proteins. This may be interpreted by the gradual change in conformation of polypeptide chains with the increase in pH, during which process the tyrosines become exposed to alkali. as they are released from the interior. This results in the overlap of pJ('s of the differently ionizing tyrosines , A related phenomenon was found by DONOVAN17 with aldolase. The irreversibility of titration curves suggests that at alkaline pH's in addition to the ionization of tyrosines, the organized structure of the protein becomes destroyed too. As a consequence, the red shift due to the native structure disappears, so that the difference in absorption caused by the phenolic ionization is lowered. This is why the real number of tyrosines can be obtained only if this denaturation blue shift is taken into account, i.e. if a denatured protein solut ion is used as reference. The distribution of tyrosines was studied both by the solvent perturbation method and by iodination of tyrosines. On the bas is of the diiodotyrosine formed , without previous denaturation, at least 35 and 20 % of the residues are inaccessible in glyceraldehyde-j-phosphate dehydrogenase and lactate dehydrogenase respectively. This might suggest that the interior parts of the proteins examined represent at least 35 and 20% of the whole molecule. Biocb im , Biophys. A eta, no (1955) 484-495

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494

The rest of the tyrosines is apparently in two different conditions. From the solvent perturbation data it appears that not more than 4S and 5S% of tyrosines is on the surfaces of the glyceraldehyde-j-phosphate dehydrogenase and lactate dehydrogenase respectively. On the other hand, about 35 and 50% of the tyrosines react instantaneously with iodine; consequently these residues are situated on the surface. The difference between the solvent perturbation and iodination data may be due to the possibility that the perturbing agent, ethylene glycol, is able to reach not only the accessible tyrosines, but some of the tyrosines partly embedded in the hydrophorbic interior. When the results obtained with the different methods are compared, complete agreement cannot be expected. The structure of a protein must not be regarded as a rigid network of polypeptide chains. Consequently the various reagents, according to their different sizes and reactivities, may penetrate to different extents into the threedimensional bulk of the molecule. In this way successive layers can be distinguished from the surface to the center of the molecule when different reagents are applied. On this basis 20-30 % and 30-35 % of the tyrosines are located in an intermediate position in glyceraldehyde-g-phosphate dehydrogenase and lactate dehydrogenase respectively. In FISCHER'S calculations'", based on the amino acid composition of the glyceraldehyde-j-phosphate, the internal area is about 50% of the whole molecule. Our perturbation results agree approximately with this estimate.

ACKNOWLEDGEMENTS

The authors are indebted to Dr. F. B. STRAUB for valuable discussion, and to Mrs. P. T6TH and Miss E. TENK for their skillful technical assistance. REFERENCES 1 P. Er.om, Biochim, Biophys, Acta, 44 (1960) 650. 2 P. EUiDI, Acta Physiol, Acad, Sci. Humg., 20 (1961) 311. 3 P. ELiiDI, G. JtCSAl AND P. T6TH, Acta Pbysiol, Acad. Sci. Hung., 23 (1963) 87. 4 1. J. HARRIS, in T. W. GOODWIN, J.1. HARRIS A!'!D B. S. HARTLEY, Structure and Activity of Enzymes, Academic Press, London-New York, 1964, p. 97. 5 L. BENEY, Biochim: Biophys. Acta, in preparation. 6 P. ELiiDI AND E. SZORENYl, Acta Phvsiol. Acad, Sci. Hung., 9 (1956) 339. 7 G. JECSAI, Acta Pbysiol. Acad. Sci.,Hung., 20 (1961) 339. 8 J. HERSKOVITS AND M. LASKOWSKI, j-, j. Bioi. Chem., 237 (1962) 2480. 9 W. L. HUGHES, JR. AND R. J. STREASSLE, j. Am. Chem, Soc., 72 (1950) 425. 10 P. Er.onr, Acta Physiol. Acad, Sci. Hung., 13 (195 8) 199. II A. PESCE, R. H. McKAY, F. R. STOLZENBACH, R. D. CAHN AND N. O. KAPLAN, j. Bioi. Chem., 239 (1964) 1753· . 12 A. N. Gr.AZER AND E. L. SMITH, j. Biol. cs-«, 236 (1961) 294 2. 13 A. N. GLAZER AND E. L. SMITH, j. BioI. Chem., 235 (1960) PC 43. 14 J. W. DONOVAN, M. LASKOWSKI, JR. AND H. A. SCHERAGA, J. Am. Chem, Soo., 83 (1961) 2686. 15 J. T. EDSALL, in G. N. RAMACHANDRAN, Aspects of Protein Structure, Academic Press, LondonNew York, 1963, p. 179. 16 D. B. WET LAUFER, Aduan, Protein Chem., 17 (1962) 303. 17 J. W. DONOVAN, Biochemistry, 3 (1964) 67. 18 R. B. MARTIN AND J. T. EDSALL, Bull. Soc. Chim. BioI., 40 (1958) 1763. 19 C. Y. eRA AND H. A. SCHRRAGA, Biochem, Biophys, Res. Commun., 5 (1961) 67. 20 D. SHUGAR, Biochem, j., 52 (1952) 142.

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DIFFERENCE SPECTROPHOTOMETR Y ON DEHYDROGENASES 21 22 23 24 25

C. T ANFORD, J . H AUENSTEIN AND D. G. RAN D,]. A m . Chem.,Soc., 77 (1956 ) 6 4 10 . H. J. F ISCHER, Proc. N atl. Ac ad . Sci . U.S ., 52 (19 64) 12 85 . C. L. GEMILL, Arch. B iochem: B iopbys ., 63 (195 6) 177 . C. L . GEM ILL, Arc h. Biocbem. Biopbys., 63 (19 56) 192 . L. G RUEN, M . LASKOWSKI, JR. AND H. A. SCHERAGA, j. Bioi. cu«, 234 (1959) 2050.

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