ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 1, November,
AND BIOPHYSICS 82-89, 1978
El ectrostatic and Conformational Effects on the Reaction of Thiol Groups of Calf Thymus Histone H3 with 5,5’-Dithiobis(2-nitrobenzoic acid) JAUME Instituto
PALAU
AND
JOAN RAMON
DABAN
de Biologia Fundamental, Uniuersidad Autbnoma de Barcelona and Consejo Superior Investigaciones Cientificas, Avenida San Antonio M” Claret, 171.Barcelona-26, Spain Received January
de
25, 1978; revised May 12, 1978
The presence of highly basic proteins (histones or protamines), causes an increase in the rate of the reaction of 5,5’-dithiobis(2-nitrobenzoic acid) (Nbsz) with the tripeptide model glutathione. This effect is explained by considering that polycationic molecules, such as histones or protamines, can attract the negatively charged reacting molecules, thus producing a catalytic effect. This effect disappears at high ionic strength due to a shielding of the charges. Urea causes a shift to the K ~(~ee)vs. pH curve for the histone H3-Nbsn reaction. This shift (2.1 units of pH for 8 M urea) indicates that urea denatures, at least to some extent, the tertiary structure of the microenvironments containing cysteine of histone H3, but it is unable to eliminate an unspecific electrostatic effect (similar to that caused by polycations in the GSH-Nbsz reaction), which also contributes to the increase of the reaction rate. Combined effects of urea and ionic strength on the reaction of GSH and of histone H3 with Nbsz gives rise to shifts of both curves of Kt,.,,, vs. pH, approaching one to the other very closely. This is interpreted as due to the appearance of shielding effects on the electrostatic charges of the histone, and also of the small molecules. The greater efficiency of guanidine hydrochloride, compared to that of urea, in causing a shift of the rate constant curve of hi&one H3 is interpreted as due to a combined effect of denaturation and electrostatic shielding in the case of guanidine hydrochloride.
Histone H3 is one of the five main fractions of the basic proteins found in association with DNA in somatic. cells of eukaryotes (1). The primary structure of calf thymus histone H3 (135 residues) is known and it indicates the presence of two cysteine residues at positions 96 and 110 of the molecule (2). Palau and Padros detected by epr spectroscopy the presence of crevices containing cysteine in the tertiary structure of calf thymus histone H3 (3) and, in a previous paper (4), the reaction of 5,5’-dithiobis(2-nitrobenzoic acid) with thiol groups of this protein was studied using the tripeptide glutathione and’ 2-mercaptoethanol as models. It was found that, under several conditions of pH, ionic strength, and temperature, histone ,H3 reacts very fast with Nbsp,l as compared with the models. This observed super-reactivity is completely lost in the presence of guanidine
hydrochloride but not in the presence of urea at pH near neutral. Evidence was found suggesting that the reaction is dependent on the nucleophile -S- concentration and that the thiol groups of histone H3 present an anomalous pKatapp) estimated as being 4.2. In the present work, we try to get a deeper insight into the mechanism of the reaction in order to clarify which is the real contribution of the tertiary structure causing the effect of super-reactivity, and which is the role, if any, of the positive charges of the histone in producing this phenomenon. Our studies provide us with some data which permit a better understanding on the behavior of the reacting thiol groups and the stability of the tertiary structure of histone H3 against denaturants.
1 Abbreviation zoic acid).
Chemicals. Nbsn was purchased from Aldrich, bovine pancreatic ribonuclease A from Calbiochem,
MATERIALS
used: Nbsp, 5,5’-dithiobis(2nitroben82
0003-9861/78/1911-0082$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
METHODS
HISTONE
H3 THIOL
GROUPS-5,5’-DITHIOBIS(2-NITROBENZOIC
horse cytochrome c from Fluka, and sodium p-chloromercuribenzoate from Cambrian. All other chemicals were supplied by Merck. Histones. Calf thymus histone H3 was prepared according to method 1 of Johns (5), using a modified procedure currently used in our laboratory (4). The purity of this histone was checked by amino acid and electrophoretic analyses. Amino acid analysis was carried out by using a Beckman M-119 Autoanalyzer. Electrophoresis was carried out according to the method of Panyim and Chalkley (6) by using polyacrylamide gels containing 2.5 M urea. Quantitation of the bands was carried out following the method of Johns (7). The H3 samples showed two bands in the monomer region (which amounted 83% of the stained material) and two bands in the dimer region (which amounted 17%). The slow band of the monomer region corresponds to the reduced form (8) and amounted to 63% of the stained material. This value is roughly in agreement with the number of reacting thiol groups per histone H3 molecule found in the samples prepared in our laboratory (4). Calf thymus histone Hl was prepared following method 1 of Johns (5) as modified by Palau et al. (9), and the protein was purified by CM-cellulose chromatography (5). A mixture of calf thymus histones H4 and H2A was prepared according to method 1 of Johns (5). Histone H4 was separated from the mixture by using the method of Phillips and Johns (10). The purity of histones Hl and H4 was checked by electrophoresis (6). Protein solutions. Solutions were prepared by dissolving the protein samples into the appropriate solvents, and the concentration was obtained directly from the weight of the samples and the volume of the solutions. The molecular weights taken for the proteins were: 2.2 X 104, 1.5 X lo4 and 1.1 X lo4 for calf thymus histone HI, H3, and H4, respectively (l), 1.4 x 10” for bovine pancreatic ribonuclease (ll), 1.3 x lo4 for horse cytochrome c (12), and 4.2 X lo3 for salmon protamine (13). Kinetics. The reaction of Nbsz with thiol groups of glutathione, 2-mercaptoethanol, and calf thymus histone H3 was followed by measuring the increase of absorbance at 412 mn (14) in a Zeiss recording spectrophotometer model DMR-21. The temperature was 25 f 0.5’ C. The reaction was studied at different pH and, for this purpose, a standard curve of the molar absorption coefficient at 412 nm of the colored product of the reaction (3-carboxylate-4-nitrothiophenolate) us. pH was used (4). In all experiments, the concentration of reacting thiol groups was approximately 5 x 10e5 M and the Nbss concentration was 7.8 x 1O-5 M. Second order rate constants, Ko(~~~),were calculated by using the current expressions employed in a preceding paper (4). The calculated values for histone H3 decrease along time, thus indicating that the dependence of the reaction rate with the concentration of thiol groups and of Nbsn, corresponds to an equation
ACID)
REACTIONS
83
more complex than that for a simple second order reaction. The calculated Kzcapp)for GSH also decrease along time in the experiments in which either basic proteins or urea-O.5 M NaCl (at low pH) are present. The values of K2,app) were taken by routine as an average of two or three calculations made within the fust minute of the reaction, inasmuch as, under a number of conditions, the reaction goes to completion in few minutes. For reactions carried out in the presence of urea at low pH, the final absorption at 412 nm was less than the expected one. This decrease is interpreted as due to the reaction of the thiol groups of histone H3, and/or 3-carboxylate-4nitrothiophenolate, with the cyanate present in urea solutions (15). Freshly prepared urea solutions were used and, for our purposes, it was assumed that the reaction with cyanate is negligible during the first minute in which the main reaction takes place. Values of Kzcspp)were computed from spectral data of this first minute and, in order to make calculations, the final absorption at 412 nm, corresponding to a reacting solution of histone H3 of the same concentration but without urea, was ordinarily used. In some cases, the final absorption at 412 nm of solutions containing urea was also used for calculations, but no relevant deviation for Kpcapp)was found. Theoretical curves of log Kzc,,,, us. pH (full or dotted lines in Fig. 3,4, and 6) were calculated according to the equation derived from current treatment of reactions whose rate is dependent on the ionization of a given species (4, 16). In some experiments, thiol groups of calf thymus histone H3 dissolved in 2.4 mM sodium phosphate, pH 7.0, were blocked with the required amount of p-hydroxymercuribenzoate according to the method of Boyer (17). It was found that the reaction product does not further react with Nbsn, thus indicating that, after the reaction of histone H3 with p-hydroxymercuribenzoate, the thiol groups of the protein become blocked. RESULTS
AND
Effect of Ionic Strength GSH with Nbsz
DISCUSSION
on the Reaction of
It was previously found (4) that a rise in the concentration of sodium phosphate from 2.4 to 24.0 nrru, between pH 5.5 and 6.7, produces a 5-fold increase of K2(appjfor the reaction of GSH with Nbsz. In Fig. 1, a detailed study of the effect of ionic strength (NaCl, pH 6.0) on the rate of that reaction is presented. A plausible explanation of the observed increment of the reaction rate can be given by considering the nucleophilic character of the reaction (4) and the electronegative nature of both GSH and Nbsz.
84
PALAU
AND
DABAN TABLE I ENHANCEMENT BY HISTONES AND OTHER BASIC PROTEINS OF THE RATE OF THE REACTION OF GSH WITH Nbsz
I 0
1 10
20
3.0
t&Cl
I4
FIG. 1. Concentration effect of NaCl on the reaction of GSH with Nbs*. The concentration of sodium phosphate in all the solutions was 2.4 mre, pH 6.0. Temperature, 25” C. Kztspp) values are plotted in a logarithmic scale.
In this sense, a raising of ionic strength would favor the ionization of the SH groups (by decreasing the activity coefficients) and also would decrease the electrostatic repulsion between GSH and Nbsz due to a shielding effect. In agreement with our view, several studies have shown that the electrostatic charge of reacting molecules has an effect on the rate of the reaction (18, 19), this effect being decreased by raising the ionic strength (19).
Protein”
pH
Protein concentration (Mb
(,Ifwg-,)
Histone Hl Histone H4 Histone H3 (SH groups blocked)* Protamine Ribonuclease Cytochrome c
6.6 6.5 6.5 6.5
5.0 x lo-” 5.0 x lo+ 5.0 x w
4.0 1.8 1.6 3.1
6.6 6.6 6.6
1.1 x 1o-4 5.0 x 1o-5 7.0 x w
2.9 x lo5 8.1 x lo3 4.6 x lo3
Histone HI Protamine
4.6 4.6 4.9
5.0 x lo-” 1.1 x w4
AND BIOPHYSICS 82-89, 1978
El ectrostatic and Conformational Effects on the Reaction of Thiol Groups of Calf Thymus Histone H3 with 5,5’-Dithiobis(2-nitrobenzoic acid) JAUME Instituto
PALAU
AND
JOAN RAMON
DABAN
de Biologia Fundamental, Uniuersidad Autbnoma de Barcelona and Consejo Superior Investigaciones Cientificas, Avenida San Antonio M” Claret, 171.Barcelona-26, Spain Received January
de
25, 1978; revised May 12, 1978
The presence of highly basic proteins (histones or protamines), causes an increase in the rate of the reaction of 5,5’-dithiobis(2-nitrobenzoic acid) (Nbsz) with the tripeptide model glutathione. This effect is explained by considering that polycationic molecules, such as histones or protamines, can attract the negatively charged reacting molecules, thus producing a catalytic effect. This effect disappears at high ionic strength due to a shielding of the charges. Urea causes a shift to the K ~(~ee)vs. pH curve for the histone H3-Nbsn reaction. This shift (2.1 units of pH for 8 M urea) indicates that urea denatures, at least to some extent, the tertiary structure of the microenvironments containing cysteine of histone H3, but it is unable to eliminate an unspecific electrostatic effect (similar to that caused by polycations in the GSH-Nbsz reaction), which also contributes to the increase of the reaction rate. Combined effects of urea and ionic strength on the reaction of GSH and of histone H3 with Nbsz gives rise to shifts of both curves of Kt,.,,, vs. pH, approaching one to the other very closely. This is interpreted as due to the appearance of shielding effects on the electrostatic charges of the histone, and also of the small molecules. The greater efficiency of guanidine hydrochloride, compared to that of urea, in causing a shift of the rate constant curve of hi&one H3 is interpreted as due to a combined effect of denaturation and electrostatic shielding in the case of guanidine hydrochloride.
Histone H3 is one of the five main fractions of the basic proteins found in association with DNA in somatic. cells of eukaryotes (1). The primary structure of calf thymus histone H3 (135 residues) is known and it indicates the presence of two cysteine residues at positions 96 and 110 of the molecule (2). Palau and Padros detected by epr spectroscopy the presence of crevices containing cysteine in the tertiary structure of calf thymus histone H3 (3) and, in a previous paper (4), the reaction of 5,5’-dithiobis(2-nitrobenzoic acid) with thiol groups of this protein was studied using the tripeptide glutathione and’ 2-mercaptoethanol as models. It was found that, under several conditions of pH, ionic strength, and temperature, histone ,H3 reacts very fast with Nbsp,l as compared with the models. This observed super-reactivity is completely lost in the presence of guanidine
hydrochloride but not in the presence of urea at pH near neutral. Evidence was found suggesting that the reaction is dependent on the nucleophile -S- concentration and that the thiol groups of histone H3 present an anomalous pKatapp) estimated as being 4.2. In the present work, we try to get a deeper insight into the mechanism of the reaction in order to clarify which is the real contribution of the tertiary structure causing the effect of super-reactivity, and which is the role, if any, of the positive charges of the histone in producing this phenomenon. Our studies provide us with some data which permit a better understanding on the behavior of the reacting thiol groups and the stability of the tertiary structure of histone H3 against denaturants.
1 Abbreviation zoic acid).
Chemicals. Nbsn was purchased from Aldrich, bovine pancreatic ribonuclease A from Calbiochem,
MATERIALS
used: Nbsp, 5,5’-dithiobis(2nitroben82
0003-9861/78/1911-0082$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
METHODS
HISTONE
H3 THIOL
GROUPS-5,5’-DITHIOBIS(2-NITROBENZOIC
horse cytochrome c from Fluka, and sodium p-chloromercuribenzoate from Cambrian. All other chemicals were supplied by Merck. Histones. Calf thymus histone H3 was prepared according to method 1 of Johns (5), using a modified procedure currently used in our laboratory (4). The purity of this histone was checked by amino acid and electrophoretic analyses. Amino acid analysis was carried out by using a Beckman M-119 Autoanalyzer. Electrophoresis was carried out according to the method of Panyim and Chalkley (6) by using polyacrylamide gels containing 2.5 M urea. Quantitation of the bands was carried out following the method of Johns (7). The H3 samples showed two bands in the monomer region (which amounted 83% of the stained material) and two bands in the dimer region (which amounted 17%). The slow band of the monomer region corresponds to the reduced form (8) and amounted to 63% of the stained material. This value is roughly in agreement with the number of reacting thiol groups per histone H3 molecule found in the samples prepared in our laboratory (4). Calf thymus histone Hl was prepared following method 1 of Johns (5) as modified by Palau et al. (9), and the protein was purified by CM-cellulose chromatography (5). A mixture of calf thymus histones H4 and H2A was prepared according to method 1 of Johns (5). Histone H4 was separated from the mixture by using the method of Phillips and Johns (10). The purity of histones Hl and H4 was checked by electrophoresis (6). Protein solutions. Solutions were prepared by dissolving the protein samples into the appropriate solvents, and the concentration was obtained directly from the weight of the samples and the volume of the solutions. The molecular weights taken for the proteins were: 2.2 X 104, 1.5 X lo4 and 1.1 X lo4 for calf thymus histone HI, H3, and H4, respectively (l), 1.4 x 10” for bovine pancreatic ribonuclease (ll), 1.3 x lo4 for horse cytochrome c (12), and 4.2 X lo3 for salmon protamine (13). Kinetics. The reaction of Nbsz with thiol groups of glutathione, 2-mercaptoethanol, and calf thymus histone H3 was followed by measuring the increase of absorbance at 412 mn (14) in a Zeiss recording spectrophotometer model DMR-21. The temperature was 25 f 0.5’ C. The reaction was studied at different pH and, for this purpose, a standard curve of the molar absorption coefficient at 412 nm of the colored product of the reaction (3-carboxylate-4-nitrothiophenolate) us. pH was used (4). In all experiments, the concentration of reacting thiol groups was approximately 5 x 10e5 M and the Nbss concentration was 7.8 x 1O-5 M. Second order rate constants, Ko(~~~),were calculated by using the current expressions employed in a preceding paper (4). The calculated values for histone H3 decrease along time, thus indicating that the dependence of the reaction rate with the concentration of thiol groups and of Nbsn, corresponds to an equation
ACID)
REACTIONS
83
more complex than that for a simple second order reaction. The calculated Kzcapp)for GSH also decrease along time in the experiments in which either basic proteins or urea-O.5 M NaCl (at low pH) are present. The values of K2,app) were taken by routine as an average of two or three calculations made within the fust minute of the reaction, inasmuch as, under a number of conditions, the reaction goes to completion in few minutes. For reactions carried out in the presence of urea at low pH, the final absorption at 412 nm was less than the expected one. This decrease is interpreted as due to the reaction of the thiol groups of histone H3, and/or 3-carboxylate-4nitrothiophenolate, with the cyanate present in urea solutions (15). Freshly prepared urea solutions were used and, for our purposes, it was assumed that the reaction with cyanate is negligible during the first minute in which the main reaction takes place. Values of Kzcspp)were computed from spectral data of this first minute and, in order to make calculations, the final absorption at 412 nm, corresponding to a reacting solution of histone H3 of the same concentration but without urea, was ordinarily used. In some cases, the final absorption at 412 nm of solutions containing urea was also used for calculations, but no relevant deviation for Kpcapp)was found. Theoretical curves of log Kzc,,,, us. pH (full or dotted lines in Fig. 3,4, and 6) were calculated according to the equation derived from current treatment of reactions whose rate is dependent on the ionization of a given species (4, 16). In some experiments, thiol groups of calf thymus histone H3 dissolved in 2.4 mM sodium phosphate, pH 7.0, were blocked with the required amount of p-hydroxymercuribenzoate according to the method of Boyer (17). It was found that the reaction product does not further react with Nbsn, thus indicating that, after the reaction of histone H3 with p-hydroxymercuribenzoate, the thiol groups of the protein become blocked. RESULTS
AND
Effect of Ionic Strength GSH with Nbsz
DISCUSSION
on the Reaction of
It was previously found (4) that a rise in the concentration of sodium phosphate from 2.4 to 24.0 nrru, between pH 5.5 and 6.7, produces a 5-fold increase of K2(appjfor the reaction of GSH with Nbsz. In Fig. 1, a detailed study of the effect of ionic strength (NaCl, pH 6.0) on the rate of that reaction is presented. A plausible explanation of the observed increment of the reaction rate can be given by considering the nucleophilic character of the reaction (4) and the electronegative nature of both GSH and Nbsz.
84
PALAU
AND
DABAN TABLE I ENHANCEMENT BY HISTONES AND OTHER BASIC PROTEINS OF THE RATE OF THE REACTION OF GSH WITH Nbsz
I 0
1 10
20
3.0
t&Cl
I4
FIG. 1. Concentration effect of NaCl on the reaction of GSH with Nbs*. The concentration of sodium phosphate in all the solutions was 2.4 mre, pH 6.0. Temperature, 25” C. Kztspp) values are plotted in a logarithmic scale.
In this sense, a raising of ionic strength would favor the ionization of the SH groups (by decreasing the activity coefficients) and also would decrease the electrostatic repulsion between GSH and Nbsz due to a shielding effect. In agreement with our view, several studies have shown that the electrostatic charge of reacting molecules has an effect on the rate of the reaction (18, 19), this effect being decreased by raising the ionic strength (19).
Protein”
pH
Protein concentration (Mb
(,Ifwg-,)
Histone Hl Histone H4 Histone H3 (SH groups blocked)* Protamine Ribonuclease Cytochrome c
6.6 6.5 6.5 6.5
5.0 x lo-” 5.0 x lo+ 5.0 x w
4.0 1.8 1.6 3.1
6.6 6.6 6.6
1.1 x 1o-4 5.0 x 1o-5 7.0 x w
2.9 x lo5 8.1 x lo3 4.6 x lo3
Histone HI Protamine
4.6 4.6 4.9
5.0 x lo-” 1.1 x w4
