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inter- and Intramolecular Crosslinks in Histone H3 Induced by 5,5’-Dithiobis(2-nitrobenzoic acid) J~~RCENBODE Gesellschaft

ftir Biotechnologische D-3300

Forschung, Abteilung Braunschweig-Stiickheim,

Molekularbiologie, West Germany

Mascheroder

Weg 1,

Received August 7, 1978 The primary reaction between the cysteine residues of histone H3 and dithiobis(nitrobenzoic acid) may be succeeded by thiol-disulfide interchange steps leading to intraor intermolecularly crosslinked H3 molecules. A chromatographic assay is applied to detect consecutive reactions of this type and is used to derive conditions by which they are promoted or suppressed. Elimination of the secondary interchange is shown to be essential if the kinetics of the reaction is to be interpreted.

An ever increasing number of proteins is found to contain cysteine side chains of a particular reactivity. A classical procedure to quantify these residues is their oxidation by thiol-disulfide interchange using Ellman’s reagent, (NBS),.’ One of its more recent applications concerns cysteine residues 96 and 110 in the sequence of histone H3 (1,2). During our own work on this protein (13), we observed that under certain conditions the reaction course is more complex than generally assumed, i.e., the NBS-S bond formed during the initial step may be split in a consecutive reaction. Similar observations have been published for other proteins with sterically close thiol groups (3). In the present contribution we describe the consequences of such a secondary reaction and a simple test for its detection. MATERIALS

Calf thymus and chicken blood were obtained from local slaughter houses. Thymus glands were stored frozen until to be used. ’ Abbreviations used: PhMeSOaF, phenylmethylsulfonyl fluoride; (NBS),, 5,5’-dithiobis(2-nitrobenzoic acid); NBS-, 5-thio-2-nitrobenzoic acid (thiophenolate anion).

From chicken erythrocytes, nuclei were released by 5% Triton and stored at 4°C after extensive washing with 0.32 M sucrose, 15 mM MgC&, and 0.2 mM PhMeSO,F. Whole histone was isolated from both sources according to (4) with a recent modification (5). Thiol contents were determined on solutions containing 1 mg protein/ml of 8 M urea, 0.1 M sodium phosphate, pH 7 (AzT6 = 0.42), after addition of WB% (41s = 0.21 for chicken histones and 0.43 for calf histones). Using an extinction coefficient for NBS of 13;600 (6) these data are in excellent agreement with theory indicating one (chicken) or two (calf) thiol groups per 65,000 daltons of whole histone protein. Kinetic runs were performed on acidextracted histones after a thorough reconstitution (dialysis via acetylated membranes (7) against 0.1 M sodium phosphate, 2 M NaCI, pH 7 or 5.5) and were found to be identical to the respective runs on histones which had been freshly extracted from chromatin by this buffer (13). RESULTS AND DISCUSSION

In an effort to isolate the H3 fraction from histone mixtures of various origins by af465

0003-2697/79/060465-05$02.00/O Copyright Q 1979 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

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finity chromatography on (NBS),-Sepharose (8) we have observed a particular complication with the H3 from calf thymus. Application of a whole histone mixture to the column led to a strongly colored effluent, the A,,, value of which indicated that all H3 was initially coupled to the solid in accord with the simple Scheme 1 or the more detailed formulas given by Lin and Foster (8). During the elution of an asymmetric protein peak, however, the adsorbent itself developed a yellow color. As the estimated ligand density of the Sepharose adsorbent should allow the attachment of only one of the two H3 cysteines, this observation is indicative of secondary interchange reaction via the other cysteine which is present in the same carboxy-terminal segment of H3 (9) (Scheme 2). That decoupling of H3 had occurred quantitatively could subsequently be verified by the failure of thiol reagents to release any further protein from a so-treated column. One of the major applications of (NBS), has been the study of individual cysteine reactivities in proteins. Thiol-disulfide interchange mechanisms of the type described might seriously affect the interpretation of the respective time courses, however, and it is highly desirable to apply a simple criterion which is suited to prove or rule out these complications. While the

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reaction kinetics could be used to detect a consecutive removal of NBS groups, revealing rates which are independent of the reagent concentration (lo), this observation can also be interpreted in different ways (2). We therefore devised a simple chromatographic assay which is based upon the precise correspondence between the chromophoric NBS--anion set free by the primary reaction and its protein-linked counterpart. This correspondence is lost by secondary SH-SS interchange steps occurring in solution as well as in the solid phase (see Schemes 1 and 2). Analysis of the Overall Reaction between Calf Thymus Histone H3 and (NBS),

We have recently derived structural aspects of histone complexes and nucleosomes from the accessibility of cysteine side chains toward (NBS), (13). In typical runs, the time course of reactions between 50 PM solutions of histone H3 (present in a whole histone complex) and varying amounts of (NBS), was monitored until the reaction was complete as judged from a constant A,,, value. In order to determine the ratio of primary and secondary reaction of (NBS)2 under these conditions, a 500~~1 aliquot of the reaction mixture was then passed through a column prepared from DEAE-cellulose in a Pasteur pipet which retained excess (NBS), and NBS- anions. The applied protein could be recovered from the colorless effluents and was submitted to a spectrophotometric quantification of bound NBS groups after treatment with 10 mM dithiothreitol. Comparison of this value and NBS- freed during the initial reaction yielded the “recovery” of H3-bound NBS groups in percentage of

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FIG. 1. Disulfide-bond formation in whole histone mediated by different amounts of (NBS),. Whole histone (50 pM) was incubated with (NBS), at different molar ratios and A,,,’ values were monitored after 24 h. Reaction mixtures were then passed over analytical DEAE-cellulose columns (0.4 X 6 cm) by consecutive washings with 800 ~1 of the reaction buffer, 1 ml 0.1 M sodium phosphate, and 8 M urea, pH 7. About 70% of applied protein was recovered from the colorless effluents (fluorescamine test) which were then adjusted to 10 mM dithiothreitol to quantify protein-bound NBS spectrophotometrically (Adz** values, obtained after correction for the protein loss). Recoveries (A,,,2. 100/A,,,l) smaller than 100% are indicative of disullide-bond formation which is seen to depend both on the histone used and the respective medium. Solid symbols are derived from calf thymus histones and correspond to 0.1 M sodium phosphate buffer, pH 7, in the presence of 0.1 M NaCl (w), 0.5 M NaCl(O), 1 M NaCl (+), 4 M urea (A). and 8 M urea (V). Open symbols (0) were obtained from chicken erythrocyte histones in 0.1 M sodium phosphate, 0.5 M NaCl, pH 7.

the theoretical value, which in Fig. 1 is plotted as a function of the reagent excess. At high molar ratios of (NBS), to histone H3 (>20), the majority of cysteines is blocked before an action upon a neighboring S-NBS bond occurs and no serious complication is anticipated if the kinetics is treated in a straightforward manner. At low (NB$-H3 ratios, however, secondary interchange (Scheme 2) becomes the dominant pathway leading to low “recoveries.” This should also hold for the situation created on NBS-Sepharose (see above), where a ratio of about 1:l of (NBS), to histone H3 is to be expected. Figure 1 further shows that reaction of (NBS), with denatured histones obviously promotes a Cys + Cys-NBS contact due to a gain in segmental flexibility.

Dimerization

Reactions

Although it is likely that an intramolecular interchange reaction between sterically close cysteines is favored over an intermolecular pathway (i.e., polymerization) such a mechanism cannot be ruled out by these experiments. We therefore extended our studies to the H3 fraction from chicken erythrocytes where-due to the presence of a single cysteine (Cys llO)-only a dimerization can occur. Figure 1 indicates that such a process is not apparent at a high enough (NBS)2 excess but it can clearly be provoked at molar ratios of (NBS)z-H3 equal to or smaller than 1. In Fig. 2 we have used a second order kinetic plot to depict the apparent time courses of the reaction between erythrocyte

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FIG. 2. Second-order kinetic plot for the reaction of chicken erythrocyte whole histone and (NBS),. Whole histone (25 PM H3) was reconstituted by dialysis against 0.1 M sodium phosphate, 2 M NaCl, pH 5.5. The reaction was started by addition of (NBS), to 1.43, 1.9.4.6, or 8.8 x lo+ M and followed by the increase of absorbance at 412 nm. c *O,ca”, Initial concentrations of (NBS), and histone H3; cx (= cB”.At/ A,), concentration of NBS1 at time t.

histone H3 and (NBS),. In this representation a dimerization reaction should be detectable by a deviation from linearity. We found that the reaction at pH 5.5 can be described by a single second-order rate constant even at a moderate excess of (NBS)2 (NBS,:H3 2 1.9). Below this value

a characteristic upward curvature is seen as the reaction proceeds, which shows that under these conditions secondary dimerization reactions are important. The deviation may be explained by an underestimation of the actual reagent concentration which in fact comprises (NBS), and H3-NBS.

FIG. 3. Dimerization between H3-NBS and H3. Whole histone from chicken erythrocytes was reacted with 27 mol (NBS)dmol H3 and purified by anion-exchange chromatography (see Fig. 1). An equal part of unmodified whole histone was then added to yield a final concentration of 50 f.cM (H3 + H3-NBS). (A) Dimerization reaction in 0.1 M sodium phosphate, pH 7, quantified by the increase of Ad,*. (B) Dimer formation (%) measured after 4 days in 0.1 M sodium phosphate media, pH 7, containing varying amounts of NaCl.

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Histone H3 dimers have attracted considerable attention in the past as it appeared conceivable that they add stabilization to the chromosome substructure (11). A dimerization reaction induced by (NBS), can conveniently be followed spectrophotometrically and is of potential use for a controlled synthesis of this species in vitro. We therefore investigated the conditions leading to an optimum dimer yield (Fig. 3). At 2 M NaCl, histone conformations are assumed to resemble those in chromatin (12) and we could show that dimerization proceeds to only 23% even after 4 days of incubation. We therefore suggest that in the native state thiols are buried in the interior of a whole histone complex. Lowering the ionic strength leads to a progressive exposure of the cysteine side chains and if NaCl is omitted from the buffer, dimerization is almost complete within several hours. ACKNOWLEDGMENTS I am very grateful to Professor K. G. Wagner, Dipl. Chem. E. Wingender and Dipl. Chem. U.D. Standt for their interest and many stimulating discussions. We also thank Mrs. R. Jahne and Miss M. Kuntz for their help and the Gefliigelzucht R. Bordan for providing facilities for the collection of chicken blood. This work

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was supported by the Deutsche Forschungsgemeinschaft (Wa 91) and the Fonds der Chemischen Industrie.

REFERENCES 1. Hyde, J. E., and Walker, I. 0. (1974) Nucleic Acids Res. 1, 203-215. 2. Palau, J., and Daban, J. R. (1974) Eur. J. Biochem. 49, 151-l-56. 3. Rippa, M., Signorini, M., Pernici, A., and Dallocchio, F. (1978)Arch. Biochem. Biophys. 186, 406-410. 4. Kobayashi, Y., and Iwai, K. (197O)J. Biochem. 67, 465-472. 5. Bode, J., and Wagner, K. G. (1975) Biochem. Biophys.

Res. Commun.

62, 868-876.

6. Silverstein, R. M. (1975) Anal. Biochem. 63, 281-282. 7. Craig, L. C. (1967) in “Methods in Enzymology” (Hirs, C. H. W., ed.), Vol. 11, pp. 870-905, Academic Press, New York. 8. Lin, L. J., and Foster, J. F. (1975)Ana/. Eiochem. 63, 485-490. 9. DeLange, R. J., Hooper, J. A., and Smith, E. L. (1972) Proc. Nar. Acad. Sci. USA 69,882-884. 10. Birkett, D. J. (1973) Mol. Pharmacol. 9,209-218. 11. Camerini-Otero, R. D., and Felsenfeld, G. (1977) Proc.

Nat.

Acad.

Sci.

USA

74, 5519-5523.

12. Kornberg, R. D. (1977) Annu. Rev. Biochem. 46, 931-954. 13. Bode, J. and Standt, U. D. (1978) Z. Naturforsch. 33c, 884-890.