Interactions of an azomercurial with proteins

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

63, 77-86 (1956)

Interactions of an Azomercurial with Proteins Myer G. Horowitz1 and Irving M. Klotz From the Department

of Chemistry, Northwestern Evanston, Illinois

University,

Received December 5, 1955 INTRODUCTION

The use of organic mercurials for the quantitative estimation of thiol groups in proteins has become well established (l-9). Various procedures, involving titrations, shifts in ultraviolet spectra or distribution between phases, have been devised. It has seemed to us that a most convenient approach might be based on a colored mercurial (4, 6), so that interaction with sulfhydryl groups could be followed by shifts in visible absorption spectra, or by changes in optical density as the bound mercurial is concentrated in the protein-containing phase. In either event, a colored mercurial must be a moderately large molecule, which might be bound to the protein at sites other t’han the sulfhydryl group. It seemed desirable, therefore, to make a thorough study of the interaction of a simple organic mercurial, 4-(p-dimethylaminobenseneazo) phenylmercuric acetate, with several proteins under a variet’y of conditions. The spectrum of this azomercurial was shifted appreciably in the presence of proteins known to have sulfhydryl groups. Likewise, cysteine or glutathione produced minor effects on the spectrum. Nevertheless, these shifts were too small to provide the basis of a precise and sensitive method of sulfhydryl analysis. On the other hand, the uptake of the azomercurial by protein sulfhydryl groups is very strong even at very low dye concentrations, and since the molecular extinction coefficient of the dye is very high, it is possible to follow changes of concentration with high precision. 1 Fellow of the National Foundation torate Fellow of the National Institutes Present address: The Jewish Hospital

for Infantile Paralysis, 1952-54; Postdocof Health, Public Health Service, 1954-55. Association, Cincinnati 29, Ohio.

77

78

MYER G. HOROWITZ AND IRVING M. KLOTZ EXPERIMENTAL

Reagents p-Aminophenylmercuric acetate was prepared from redistilled aniline and mercuric acetate according to the procedure of Dimroth (10). It was then diazotized with sodium nitrite in 50% acetic acid (11,12), and the diazonium salt was filtered and coupled with freshly distilled dimethylaniline in the cold. The product, 4-(pdimethylaminobenzeneazo)phenylmercuric acetate, (I), was extracted with hot n-amyl alcohol and purified three times by dissolution in glacial acetic acid followed by addition of 4 vol. of n-amyl alcohol. M.p. 214-216’ (uncorr.); lit. (ll), 215’ (uncorr.). Anal.: Calcd. for ClsHlrOaNaHg: C 39.71%, H 3.54Qj N 8.68%; found: C 39.660/ H 3.75%, N 8.67%. Crystallized bovine serum albumin, crystallized @-lactoglobulin, and bovine r-globulin were purchased from Armour and Co., crystallized ovalbumin from Worthington Co. A sample of human serum albumin was obtained from Drs. P. H. Bell and R. 0. Roblin, Jr., of the American Cyanamid Co. Iodinated bovine albumin was prepared by Dr. W. L. Riedeman following the procedure of Hughes and Straessle (13). Glutathione and cysteine hydrochloride were products of Schwarz Laboratories, glycine and n-heptanol of the Matheson Company, and guanidine hydrochloride of Eastman Kodak Company. All inorganic chemicals were C.P. grade. (CH&Na-N=Na-Hg(OOCCH8)

. Absorption

Spectra

The absorption of light was measured with the Beckman model DU spectrophotometer at approximately 25°C. One-centimeter cells were used.

Partition

Analysis

The binding of azomercurial by protein was measured by following the removal of dye from an immiscible nonaqueous solvent in equilibrium with the proteincontaining aqueous phase. The principle of this method has been used previously with organic mercurials (5, 6) as well as with other organic molecules (14). In this investigation the aqueous phase consisted, in general, of a 0.1 M glycine buffer, for reasons to be mentioned later. A variety of organic solvents were examined, but heptanol was chosen since it provided a convenient distribution coefficient combined with a small phototropic effect on the dye (15). Each phase was saturated with the other before use. Several hundred milliliters of glycine buffer was shaken with 10 ml. of heptanol for 3-4 hr. in a water bath adjusted to the temperature to be used in the dye equilibration, and suspended alcohol droplets were removed thereafter by centrifugation at the same temperature. A similar procedure was used to saturate the heptanol with aqueous buffer solution. Temperature control was necessary during these equilibrations since the alcohol and water solubilities are sensitive to temperature changes.

PROTEIN-AZOMERCURIAL

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INTERACTION

An appropriate quantity of azomecurial was then dissolved in the alcohol, the container being covered with aluminum foil to exclude light and thereby to prevent the trans-cis conversion (15), A stock protein solution in aqueous buffer was then prepared. Thereafter, a series of flasks were assembled containing 98 ml. of aqueous phase, with protein dissolved in it, and 2 ml. of heptanol with different quantities of dissolved azomercurial. Each flask was covered with aluminum foil, placed in a water bath (shielded from room light), and shaken gently for about 4 hr. This period of shaking was found adequate for the attainment of equilibrium, and the rate was sufficiently slow to avoid any appreciable denaturation of the protein. The 98:2 ratio of volumes of aqueous to alcohol phase was used to counterbalance the high value of the partition coefficient in favor of the organic solvent. Partition coefficients were measured in each experiment by having control flasks with protein deleted from the aqueous phase. At the completion of the equilibration period, an aliquot of the aqueous phase was pipetted off and centrifuged at a controlled temperature, and its optical density (at 460 rnp) was read in the Beckman spectrophotometer. A similar procedure was followed with the alcohol layer. In t,he latter case, however, it was necessary to keep in mind that readings of optical density (taken at 414 11111) increase with time up to 5-10% above the initial reading, due to the phototropic properties of this azomercurial dye (15). From the decrease in optical density of the alcohol layer, one can compute how much dye must have been taken up by the protein-containing aqueous phase. A certain portion of the dye would be in the aqueous phase even in the absence of protein, and this quantity is measured by the cont,rol flasks. The excess of dye dissolved in the aqueous phase in the presence of protein is taken as the amount bound by the protein.

RESULTS Spectra

AND DISCUSSION

of Complexes

4-(p-Dimethylaminobenzeneazo)phenylmercuric acetate has an absorption spectrum (Fig. 1) typical of (CHs)JV-substituted azobenzenes. The solubility of this azomercurial in water or in most buffers, however, is so small that the absorption of light cannot be measured conveniently. On the other hand, in the presence of glycine, the mercury dye will dissolve appreciably, at pH’s from 6 to 10, and shows a peak at 458 rnp and a molecular extinction coefficient of 26,000. The effect of glycine is probably due to the following subst’itution reaction: R-Hg-OOCCH3

+

H2NCH&OO-

Upon addition

--t R-Hg+-NH&H&OO-

+

CH,COO-

(I)

of cysteine or glutathione (mercaptan to dye ratio in glycine buffer pH 9.6, a small increase in absorption is observed (E becomes 27,500 at 458 mp). In the presence of bovine serum albumin, on the other hand, the absorption of the ca. 1.5) to t’he azomercurial

80

MYER

00,000

G.

HOROWITZ

AND

IRVING

M.

KLOTZ

-

e

l0,000

-

0

300

FIG. 1. Spectra of (6 X 1P M) in glycine dye and glutathione or serum albumin, 2.8 X

I 350

I 400 Wovelength,

I 450

I 500

55

mp

4-(p-dimethylaminobenzeneazo)phenylmercuric acetate (0.1 M)-acetate (0.04 M) buffer pH 9.5: (1) dye alone; (2) cysteine hydrochloride mole ratio 1: 1.5; (3) dye and bovine 10-h M.

azomercurial (Fig. 1) is reduced, the maximum becoming much flatter and broader and shifted to lower wavelength (cu. 430 mp). The opposite direction of the shift in absorption in the presence of albumin, as contrasted to glutathione, shows that the general effect of the protein on the absorption of the azobenzene molecule overwhelms that of the formation of the mercaptide bond. Other sulfhydryl-containing proteins, such as ovalbumin, @-la&oglobulin, and human serum albumin, shifted the spectrum of the azomercurial in the same manner as bovine albumin. On the other hand, the presence of bovine r-globulin was essentially without effect on the azomercurial spectrum. In principle, therefore, the visible spectrophotometric changes provide a basis for quantitative determination of protein sulfhydryl groups analogous to the ultraviolet method of Boyer (8), perhaps with the advantage that absorption of light by the protein is negligible in the visible region. In practice, however, the changes in absorption produced by the protein do not exceed 20% and thus seem too small to provide a precise method of analysis. An alternative approach would be to measure the increased optical density, due to increased solubility, when solid azomercurial dye is shaken with a solution containing protein, as contrasted to one without protein. Such an experiment was carried out with bovine albumin in 0.1 M glycine buffer pH 9.3. As anticipated, much more dye dissolved

PROTEIN-AZOMERCURIAL

81

INTERACTION

in the presence of protein, the moles of bound dye per mole protein rising rapidly (within 1 hr.) to 0.5 and slowly thereafter to 0.7 (after 44 hr.). At pH 6.9, however, the moles bound had reached only 0.4 after 50 hr. The very long times required for equilibrium make this approach unattractive. It may be possible, however, to introduce the modification of dissolving the dye in concentrated acetic acid and then adding small quantities of this solution to a buffered protein solution and to buffer. Binding

Measurements by Partition

Method

Equilibration Time. In the absence of protein, equilibrium in distribution of azomercurial between immiscible phases is reached in about 4 hr. In the presence of protein, a similar period is adequate unless mercaptide formation is a slow reaction in itself. Thus with bovine serum albumin and with P-lactoglobulin (Table I), t’he extent of binding of mercurial was the same at 8 hr. as at 4.5 hr. of equilibration. With ovalbumin, however, there is evidence (Table I) that the reaction Cth mercurial is slow. This behavior, plus the stoichiometric data to be shown below, fit the assumption that at least some of the sulfhydryl groups of ovalbumin are masked. Dependence of Partition Coeficient on pH. The distribution of the dye between n-heptanol and aqueous glycine varies with pH, and hence the partition coefficient was measured over a wide pH range. The results TABLE I Effect of Equilibration Time on the Binding

of

mw,iv~-.v=N~-~gby Protein Protein

PH

Bovine serum albumin,

6 X

10-C M &Lactoglobulin,

Ovalbumin,

6 X 10-e M

6 X 10-G M

at 25”

Aqueous free dye concentration, M x 107

Time of equilibration, hours

Moles bound dye per mole protein

9 9

13.9 13.8

4.5 8

0.670 0.675

9.6 9.6

15.4 15.6

4.5 8

1.83 1.83

9.1 9.1 9.1 9.6 9.6

13.3 9.81 7.33 7.08 3.56

4.75 8 17 4.5 8

2.48 2.72 2.86 2.53 2.74

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G.

HOROWITZ

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M.

KLOTZ

FIG. 2. Variation in the distribution of aeomercurial (I) between n-heptanol and aqueous 0.1 M glycine solution as a function of pH at 25°C.

are illustrated in Fig. 2, the partition coefficient being taken as the ratio of azomercurial concentration in the organic phase to that in the (protein-free) aqueous phase. A relatively flat curve is obtained over the pH range of 7-9.6. The rise in partition coefficient in favor of the organic phase, below pH 7, is probably due to displacement of the polar NH.GH&OOligand from the mercury [see Eq. (l)], as the zwitterion of glycine forms. The upward trend at pH 10 may mark the beginning of the displacement of NH2CH2COO- by OH- ion. In either event, removal of the polar glycinate ion from the azomercurial would tend to make the dye less soluble in water and more attracted by the organic phase. Binding by Bovine Albumin. The most detailed study of the uptake of azomercurial was made in 0.1 M aqueous glycine buffer of pH 9.6. These results are summarized in Fig. 3, where r represents the moles of bound mercury dye per mole of serum albumin. The curve rises very rapidly with increasing dye concentration until 0.66 mole of dye are bound per mole of protein; thereafter it remains flat over a 50-fold increase in concentration. The wide range of this plateau emphasizes the specificity of the interaction of mercurial with protein under these conditions. An r value of 0.66 corresponds to the sulfhydryl content of bovine serum albumin as determined by other methods (5, 16, 17). Further corroboration of the assignment of 0.66 mole to mercaptide formation

PROTEIN-AZOMERCURIAL

83

INTERACTION

3

-

.

FIG. 3. Binding of azomercurial (I) by 6 X 1OP M bovine serum albumin at pH 9.6, 0, or at pH 6.0-6.4, 0, and 25°C. in 0.1 M aqueous glycine solution; T is moles of dye bound per mole of protein.

was obtained in an experiment with iodinated bovine albumin, in which the sulfhydryl group has been oxidized. Only 0.05 mole of mercurial was bound by the iodoprotein at a dye concentration of 7 X 10e7 M. This small uptake is probably due merely to binding of mercurial by ionized phenolic groups of t,he iodotyrosine residues in the protein, whose pK lies in t’he neighborhood of 7 (18).

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HOROWITZ

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M.

KLOTZ

Perhaps the most interesting feature of Fig. 3 is the very wide range of concentration over which the plateau extends. The right-hand-side limit of this flat line was never reached because higher concentrations of free dye could not be produced. One would expect the mercurial to be bound to protein amino groups when the sulfhydryls have been saturated. The complete selectivity for, and stoichiometry with, the thiol content (Fig. 3) is probably due to the use of glycine; the amino group of NH#ZH$ZOOprobably forms a strong competing ligand for the mercury against all competitors except the protein -SH. This interpretation is substantiated by measurements of binding by bovine albumin at pH 6 (Fig. 3). Although glycine was also present in this buffer, it exists overwhelmingly in the dipolar form, H3+NCH&OO-. In contrast, the protein at pH 6 possesses a number of nonprotonated basic nitrogen atoms in its histidine residues.2 The binding of azomercuria1 (Fig. 3) jumps very sharply to a value of 0.56 for r at a dye concentration of about 1 X lo-’ M. A sharp break in binding occurs at this point, and the curve tends to become horizontal. However, in marked distinction to the behavior at pH 9.6 (Fig. 3), the curve for pH 6 shows a definite slope in the range of 1-15 X lo-’ M and acquires a very pronounced upward trend thereafter. Values of r above 3 have been measured, and the curve indicates that even greater binding might have been observed with a more soluble mercurial. Clearly such high r values must be due to binding of azomercurial by groups in addition to the thiol, presumably imidazole nitrogens. Thus at pH 6, interaction of the mercurial with thiol and basic nitrogen residues is not so sharply differentiated as at pH 9, and the T value clearly assignable to -SH groups tends to be slightly low. Hughes (5) has also mentioned that for stoichiometric reaction of -SH groups with methylmercury iodide, the pH must be above 7. Binding by p-Lactoglobulin. The uptake of azomercurial by this protein at pH 9.1-9.6 is summarized in Fig. 4. A plateau, at an r value of 1.83, extends to a free dye concentration of about 30 X lo-’ JR, and a very slight rise seems evident thereafter. The r value of 1.83 agrees with the -SH content of p-lactoglobulin determined by nonoxidative methods, but oxidation procedures tend to give higher values (8, 20-23). Binding by Ovalbumin. Measurements with an equilibration time of 2 It should also be mentioned that at pH 6, the fraction of ionized -SH groups is less than 1:lOOO that at pH 9.6. Furthermore, at the higher pH, the protein is in a more swollen, expanded configuration (19).

PROTEIN-AZOMERCURIAL

85

INTER.kCTIOK

,

I

20

I

I

60 M I IO’

I

60

FIG. 4. Binding of azomercurial (I) by 6 X 1O-6 M protein at 25°C. in 0.1 14 aqueous glycine solution: lower curve, p-lactoglobulin, pH 9.1-9.6; upper curve, ovslbumin, pH 9.1.

4.5 hr. at pH 9.1 are summarized in Fig. 4. It is apparent that two of the mercapt,an groups react very readily wit’h the mercurial. The third thiol is definitely less reactive, and t’he fourth seems t,o show a further small drop in reactivity. From these measurement,s it is not possible to determine with assurance the t’otal number of --SH groups in ovalbumin, but the results are consistent with a value of 4 (8, 16, 24). In contrast to titratjions with p-mercuribenzoat’e in which three of the four thiols seem more reactive, partition equilibria with the azomercurial indicate that t’no of the sulfhydryl groups are more accessible to the reagent. Paralleling previous observations (5, 8), we find t,he reaction with ovalbumin t’o be slow. Conclusion Wit,h a colored azomercurial it is thus possible t.o measure the thiol content of proteins by several procedures. The most precise of these is based on the partition of dye between two immiscible liquid phases. The very high optical absorption of the dye permits one to make measuremenk at very low dye concenkations (~10-~ AZ) and wibh very dilute protein solutions (6 X 10e6 M) without any interference due to absorption of light by the protein. Furthermore, t’he use of a suitable buffer, glycine in this investigation, establishes conditions for a high specificity toward thiols in the interaction of the mercurial with proteins. This method is distinctly more laborious than some other procedures

86

MYER G. HOROWITZ AND IRVING M. KLOTZ

(8, 16, 17), but it seemslikely that, as with hemoglobin (9), a combination of methods is apt to give the greatest insight into structural problems. SUMMARY

With a colored mercurial, 4-(p-dimethylaminobenzeneazo)pbenylmercurie acetate, it is possible to measure the thiol content of proteins from shifts in visible absorption spectra or from changes in optical density as mercurial is concentrated in an aqueous, protein-containing phase, The latter procedure is more precise and gives values of 0.66, 1.83, and about 4 for the number of moles of -SH per mole of bovine serum albumin, &lactoglobulin, and ovalbumin, respectively. Differences in reactivity of thiols can also be distinguished. REFERENCES 1. HELLERMAN, L., Physiol. Revs. 17, 454 (1937); Cold Spring Harbor Symposia Quant. Biol. 7, 165 (1939). 2. ANSON, M. L., J. Gen. Physiol. 24, 399 (1941). 3. SINGER, T. P., AND BARRON, E. S. G., J. Biol. Chem. 167, 241 (1945). 4. BENNETT, H. S., Anat. Record 100, 640 (1948). 5. HUGHES, W. L., JR., Cold Spring Harbor Symposia Quant Biol. 14, 79 (1949). 6. FLESCH, P., AND KUN, E., Proc Sot. Exptl. Biol Med. 74, 249 (1950). 7. BENESCH, R., AND BENESCH, R. E., Arch. Biochem. and Biophys. 38,425 (1952). 8. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 9. INGRAM, V. M., Biochem. J. 69, 653 (1955). 10. DIMROTH, O., Ber. 36, 2032 (1902). 11. JACOBS, W. A., AND HEIDELBERGER, M., J. BioZ. Chem. 20, 513 (1915). 12. BENNETT, H. S., AND YPHANTIS, D. A., J. Am. Chem. Sot. 70, 3522 (1948). 13. HUGHES, W. L., JR., AND STRAESSLE, R., J. Am. Chem. Sot. 73, 452 (1950). 14. KARUSH, F., J. Am. Chem. Sot. 73, 1246 (1951). 15. HOROWITZ, M. G., AND KLOTZ, I. M., J. Am. Chem. Sot. 77,501l (1955). 16. BENESCH, R., AND BENESCH, R. E., Arch. Biochem. 19, 35 (1948). 17. KOLTHOFF, I. M., &RICKS, W., AND MORREN, L., Anal. Chem. 26, 366 (1954). 18. KLOTZ, I. M., AND URQUHART, J. M., J. Am. Chem. Sot. 73, 3182 (1951). 19. KLOTZ, I. M., BURKHARD, R. K., AND URQUHART, J. hf., .I. Am. Chem. Sot. 74, 202 (1952). 20. GROVES, M. L., HIPP, N. J., AND MCMEEKIN, T. L., J. Am. Chem. Sot. 73,279O (1951). 21. FRAENKEL-CONRAT, J., COOK, B. B., AND MORGAN, A. F., Arch. Biochem. and Biophys. 36, 157 (1952). 22. LARSON, B. L., AND JENNESS, R., J. Am. Chem. Sot. 74, 3090 (1952). 23. CHRISTENSEN, L. K., Compt. rend. trav. lab. Carlsberg, Ser. chim. 26,37 (1962). 24. MACDONNELL, L. R., SILVA, R. B., AND FEENEY, R. E., Arch. Biochem. and Biophys. 32, 288 (1961).