Limited Proteolysis of Horse Heart Cytochrome c

Transcription

1 Eur. J. Biochem. 20 (1971) Limited Proteolysis of Horse Heart Cytochrome c Abel SCREJTER, Tzipora GOLDKORN, and Mordechai SOKOLOVSEY The Department of Biochemistry, Tel-Aviv University (Received January 23/March 23,1971) Proteolysis of cytochrome c with pepsin results in the rapid preferential cleavage of the peptide bond between residues 66 and 67. The resulting 1-66 heme peptide is biologically inactive, binds readily cyanide and carbon monoxide, is oxidized in air, but is not reduced by ascorbate. The spectrum of this heme peptide has the same maxima observed for shorter heme peptides, but the absorption coefficients of their maxima are significantly lower. It is a well known fact that most proteins in their native states are resistant to proteolysis. Rates of proteolysis, therefore, have been proposed as indicators of the degree of denaturation or extent of conformational changes in proteins [I -71. The proteolysis of horse heart cytochrome c was investigated using papain, trypsin, chymotrypsin and pepsin [8,9]. With the latter, an interesting phenomena was observed [8]: at the very early stage of peptic hydrolysis, the absorbance of the a-band decreased up to 60 /, of its initial value, followed by increases in the succeeding stages to yield a final product which had an absorbance of 80 Ol0 of the native protein. The final product [9] had an absorption spectrum closely similar to that of native cytochrome c and it was suggested that the prosthetic group together with the polypeptide chain in its immediate vicinity were not appreciably changed during the digestion. This final product is the hemepeptide isolated and characterized later [lo]. However, the nature of the changes of the a-band at the early states of proteolysis remained unanswered. This communication describes in detail the structural changes which accompany the early stages of proteolysis and the physicochemical properties of a heme peptide isolated as the main product of short term peptic hydrolysis. EXPERIMENTAL PROCEDURE Materials Commercial horse heart cytochrome c type I1 was obtained from the Sigma Chemical Company, and purified on Amberlite CG-50 [Ill. Pepsin, twice crystallized, was obtained from Worthington. All other chemicals were of the best grade available. Methods Concentrations of Native Cytochrome c were determined by the absorbance of the reduced form at 550 nm using a molar absorption coefficient of 2.77 x lo4 M-l cm-l [12]. Xpectrophotometric Experiments were carried out with a Zeiss PMQ spectrophotometer for measurements of absorbance at single wavelengths, and a Cary 15 recording spectrophotometer was employed for determination of absorption spectra. The ph of Solutions was measured on a Radiometer Model 26pH meter equipped with a glasscalomel combination electrode. Amino Acid Analyses were performed with a Beckman-Unichrome amino acid analyzer according to the procedure of Spackman et al. [13]. Samples were hydrolyzed in constant boiling HC1 in evacuated sealed tubes in the presence of 10 pl phenol, at 110 "C for 22h. Tryptophan was determined on the unhydrolyzed protein and peptides using N-bromosuccinimide [14]. Cytochrome c Activity was estimated by the method of Jacobs and Sanadi [15]. Amino Terminal Residues in the modified cytochrome c were determined both by the cyanate method [IS] and by dansylation [17]. The dansyl amino acids obtained after acid hydrolysis were detected after separation by two dimensional thin-layer chromatography on polyamide sheets [MI. The first solvent was 1.35 Ol0 formic acid and the second solvent was benzene-glacial acetic acid (9: 1, v/v). Course of Proteolysis. The course of digestion of cytochrome c (6mg/ml) with a freshly prepared solution of pepsin (0.03 mg/ml) was measured by the absorbance at 550 nm of the reduced form. Aliquots of 0.2 ml were withdrawn and diluted into 2 ml0.1m Tris-HCI ph 8, and the solution was reduced with sodium dithionite. RESULTS Peptic Digestion of Cytochrome c As shown in Figs. 1 and 2, within 2min of digestion at ph 1.8, there is a rapid change in the absorbance.

2 V01.20, No.3,1971 A. SCHEJTER, T. GOLDEORN, and M. SOEOLOVSEY I I I I 1 r Time (min) Fig. 1. Kinetics of pepsin action on ferricytochrome c at 28' C followed by measuring absorbance at 550nm after reduction with sodium dithionite. The reaction mixtures contained 5.5 mg per ml of ferricytochrome c and mg per ml of pepsin in 0.1 M acetate buffer ph 4.5 (O), 0.1 M acetate buffer ph 3.4 (0) or 0.04 M HCl ph 1.8 (A) Table I. Determination of NH,-terminal groups in pepsinmodified cytochrome c Peptic digestion of cytochrome c was carried out for 3 min at ph 1.8 and 23 "C NH,-terminal amino acids Tyrosine Aspartic acid Glutamic acid Glycine Alanine Amount moles/mole protein g0 m e 5: I I I I I I Time (rnin) Fig. 2. Kinetics of pepsin action on ferricytochrome c at 23" C, followed by measuring absorbance at (A) 418 nm, (B) 550 nm, (C) 520 nm, after reduction with sodium dithwnite. The reaction mixtures contained 7.5 mg per ml of pepsin in 0.04 M HCI, ph ' J 80 At ph 3.4 similar results are obtained though longer periods of digestion are necessary while at ph 4.5 the reaction is much slower. For routine preparation of the products, the digestion was carried out for 3 min at ph 1.8 and 23 "C. Amino Terminal Groups Since the N-terminal residue of native cytochrome c is blocked by an acetyl group, any new N-terminal residue formed must result from the proteolysis. Table 1 summarizes the results of N-terminal amino acid analyses by the cyanate and the dansyl method. Aspartic and glutamic acids (or their corresponding amides) and tyrosine are the N-terminal residues of the fragments formed. Analyses of the products at various digestion periods indicated that tyrosine was always the major N-terminal component of the peptides obtained. 28'

3 416 Limited Proteolysis of Horse Heart Cytochrolse c Eur. J. Biochem. I I I I 1 1 Fraction number Fig.4. Elution diagram of pepsin modified cytochrome c, obtained by chromatography on Amberlite Ccf-25 (2 x 20 cm) with a linear Nu+ concentration gradient. The dilute solution of Naf contained 0.02 M sodium phosphate buffer, ph 8.0, 400 ml, the concentrated solution the same buffer containing 0.6 M sodium chloride 400 ml. The dashed line represents the Na+ concentration in the eluate. Fractions collected were 3 ml. Absorbance was measured at 410 nm (0) and 220 nm) (0) Table2. Amino acid composition of cytochrome c and of the two fractions of pepsin modified cytochrome c ~~ Amount in Heme carrying Heme Amino acid Native fraction of free-fraction of cytochrome c pepsin modifled pepsin modified cytochrome c cytochrome c moles/mole Lysine Histidine Arginine o 0.9 Threonine Aspartic acid Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine o Tryptophan Total numbers of residues Yield of isolation CG-50 (Fig.4). In this case, the peak eluting before the gradient was free of the heme group, while the second fraction was eluted with 0.25 M NaCl, similar to native cytochrome c. Further attempts to purify the heme free peak were unsuccessful. Amino Acid Analyses of Digestion Products The amino acid composition of the heme free fraction, the fraction carrying the heme, and native cytochrome c, are listed in Table 2. The analyses indicate that the fist fraction is composed of roughly one third of the molecule, while the heme carrying fraction consisted of the remaining two thirds. The sum of the two fractions agreed well with the composition of the native enzyme. From the elution pattern of the heme carrying fraction by molecular sieve chromatography on Sephadex G-50 a molecular weight of about 8500 was obtained. Enzyme Activity The enzymic activity of pepsin modified cytochrome c was measured as a function of the time of proteolysis. As shown in Fig.5, the addition of a sample of the products of proteolysis to cytochrome c depleted mitochondria did not restore their respiratory function. The decrease in activity was parallel to the change in absorbance at 550 nm. When mixtures of the purified 1-66 heme peptide and the peptide were added to cytochrome c depleted mitochondria, in proportions varying from 1 : 10 to lo;l, they failed to restore the respiration of the depleted mitochondria. Spectroscopic and Chemical Properties At near neutral ph, the spectrum of the heme carrying peptide is quite similar to that of native cytochrome c (Fig.6). However, three major differences should be noticed: first, the 695 nm band [19]

4 Vol. 20, No A. SCHEJTER, T. GOLDKORN, and M. SOKOLOVSKY I I ,-" x._. - > Time (min) Fig.5. Respiratory activity of cytochrome c depleted rat liver mitochondria in the presence of exogenous horse heart cytochrome c, modified by pepsin, as a function of the time of proteolysie. The oxygen consumption was measured with an oxygenelectrode, in a reaction mixture containing 9 mg mitochondria1 protein; the composition of the medium was as given by Jacobs and Sanadi [15]. Pepsin action on ferricytochrome c was followed at 23"C, in a reaction mixture that contained 7.5 mg per ml of ferricytochrome c and mg per ml of pepsin in 0.04 M HCl, ph 1.8 I I I I I "." Wavelength (nm) Fig.7. Spectra of ferrous pepsin modified cytochrme c...., 0.1 M acetate ph 4.0; -, 0.1 M phoephate ph 7.0; 0.1 M glycine-naoh buffer ph 9.5 is absent from the oxidized form of the heme peptide ; second, the Soret band of the oxidized heme peptide is shifted by 2 nm towards the blue, when compared to the native protein; third, the absorption coefficients of the a and bands of the reduced heme peptide are distinctly lower than in native cytochrome c, but increase with increasing ph (Fig.7). The heme peptide binds readily cyanide, both in the oxidized and in the reduced states, and carbon monoxide in the reduced state. The spectra of these complexes are identical to those shown by the analogous complexes of native cytochrome c. The heme peptide is reduced by dithionite, and the reduced heme peptide is oxidized rapidly in air, at neutral ph. Ascorbate, cannot reduce the heme peptide, even at concentrations of 1 mm of the reducing agent, that are sufficient to reduce completely native cytochrome c in about 5min. This lack of reducibility is not due to immediate reoxidation by oxygen; in fact, no oxygen consumption could be detected polarographically when ascorbate was added to the 1-66 heme peptide. 0.1 " I I 1 I I Wavelength (nm) Fig.6. Visible spectra of cytochrome c and pepsin modified cytochrome c in 0.1 M Tris-HCl buffer, ph , ferrocytochrome c; ----, ferrous pepsin modified cytochrome c;..., ferric cytochrome c; -.-.-, ferric pepsin modified cytochrome c DISCUSSION The initial event in limited proteolysis of native cytochrome c must take place either at a peptide bond exposed to the solvent in a rigid compact structure, or at one exposed in some partially unfolded species. Since pepsin was employed for proteolysis, acidic media had to be used, thus converting the cytochrome c from the close crevice structure, stable at neutral ph, to the open crevice structure found at low ph [20]. Therefore the bond split is most probably of the second type.

5 418 Limited Proteolysis of Horse Heart Cytochrome c Eur. J. Biochem. The fact that high molecular weight intermediates could be isolated indicates that the initial attack occurs at a rate much greater than the subsequent cleavages. After the initial proteolytic split only two fractions are obtained with a reasonable yield : a heavy fraction, with a molecular weight of 8500, containing the prosthetic group and composed of two-thirds of the native enzyme, and a heme-free fraction, composed of one-third of the enzyme. The amino acid composition of the heme peptide fits that of the sequence 1-66,while that ofthe hemefree fraction fits the C-terminal portion beginning with residues in the region of positions 65 to 68 in the sequence of the native enzyme [21]. Horse cytochrome e contains only 2 methionyl residues at positions 65 and 80 in the primary structure [21], and since the amino acid composition indicates the presence of one methionine in each fraction (Table 2), then the cleavage must have occurred between these two residues. Furthermore, the heme- fraction contains only one tyrosyl residue, while the other three tyrosyls, 67, 74 and 97, are in the second fraction. Therefore, the cleavage had to occur between methionine 65 and tyrosine 67. Finally, tyrosine has been identified as the major new N-terminal released during the proteolysis. On the basis of this evidence we conclude that the bond glutamyl (66)-tyrosine (67) was cleaved. The presence of other N-terminals is not surprising because of the known broad specificity of pepsin [22]. If additional cleavage had occurred at the amino group of glutamic residues 66 or 69 or asparagine 70, it should yield the same peptide obtained with cleavage at tyrosine-67 but shorter in one amino acid or longer in 2-3 residues. This may explain why such residues appear as N-terminals in addition to tyrosine, while further fractionations of the heme- free fraction were unsuccessful. The very rapid limited proteolysis of cytochrome c is reminiscent of that of ribonuclease A [4]. In both cases the cleavage results with the formation of two fractions which are not separatable under normal7 conditions, i. e. gel filtration at neutral ph. In the case of cytochrome c the separation could be effected only at acidic conditions (0.01 N HC1) which is probably due to the fact that part of the native tertiary structure remains unaffected after the early proteolysis has split a single peptide bond. Separation of the two fractions was possible only by applying acidic conditions or using ion-exchange chromatography at neutral ph. However, in contrast to ribonuclease A, the derivative obtained by Limited proteolysis was inactive. Since the inactivation proceeds sincronously with the spectral changes and the release of tyrosine as N-terminal, we conclude that even though the two fractions are still held together, the bond is crucial in maintaining the biologically active conformation. Changes that occurred in this area of the protein might affect the neighboring methionyl-80 [23] which in turn will result in the disappearance of the 695 nm band and the biological activity. This assumption is strongly supported by similar evidence obtained recently from the nitration of cytochrome c which resulted in the selective nitration at tyrosine 67 [24]. Thus, the high reactivity of this residue, both in nitration and in limited proteolysis, might be due to a microenvironment which is directly related to methionyl-80, the ligand of the iron atom in native cytochrome c [23]. The existence of a spatial closeness between the tyrosyl-67 and methionyl-80 residues has been demonstrated by recent crystallographic studies [23]. The spectroscopic and chemical properties of the 1-66 heme peptide are closely similar to those of the much shorter heme-endecapeptide obtained by terminal peptic proteolysis [9]. The absence of the 695nm band is obviously due to the removal of methionine 80 [25]. The blue shift of the Soret peak indicates that the 1-66 heme peptide contains a small but distinct amount of high spin component. The ph dependence of the intensity of the 01 and /I bands of the reduced form of the 1-66 heme peptide was already observed in the heme peptide [26] and in cytochrome c carboxymethylated at the methionine 80 sulfur [27]. The high reactivity towards ligands i. e. cyanide and carbon monoxide, is a clear indication that in the 1-66 heme peptide the closed crevice structure is much weaker than in native cytochrome c.1t should be pointed out, however, that the 1-66 heme peptide is unable to mediate the oxidation of ascorbate by oxygen, a function that is readily accomplished by the smaller heme peptides [26]. With regard to this type of reaction, the heme group in the 1-66 heme peptide appears to be inaccessible to organic electron donors. Whether this inaccessibility is of a steric nature, or rather an indication of lack of electron paths from the peptide periphery to the heme, is a problem that deserves further investigation. REFERENCES 1. Rupley, J. A., in Methods in Enzymology (edited by C. H. W. Hire), Academic Press, New York 1967, Vol. XI, p Ottesen, M., Annu. Rev. Biochem. 36 (1967) Ooi, T., Rupley, J. A., and Scheraga, H. A., Biochemistry, 2 (1963) Richards. F. M.. and Vithavathil. P. J.. J. Biol. 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6 V01.20, N A. SCHEJTER, T. GOLDKORN, and M. SOKOLOVSKY Margoliash, E., and Walasek, 0. F., in Methods in Enzymology (edited by R. W. Estabrook and M. E. Pullman), Academic Press, New York 1967, Vol. X, D Margoliash, E., Frohw&, N., and Wiener, E., Bhchem. J. 71 (1959) Spackman, D: H., Stein. W. H., and Moore, S., Anal. Chem. 30 (1958) Spande, T. F., and Witkop, B., in Methods in Enzymology (edited by C. H. W. Hirs), Academic Press, New York 1967, Vol. XI, p Jacobs, E. E., and Sanadi, D. R., J. Biol. Chem. 235 (1960) Stark, G. R., and Smyth, D. G., J. Biol. Chem. 238 (1963) Gray, W. R., in Methods in Enzymology (edited by C. H. W. Hirs), Academic Press, New York 1967, Vol. XI, p Woods, K. R., and Wang, K. T., Biochim. Biophys. Ackc, 133 (1967) Schejter, A., and George, P., Biochemistry, 3 (1964) Theorell, H., and ilkesson, A., J. Amer. Chem. Soc. 63, (1941) Margoliash, E., J. Biol. Chem. 237 (1962) Smyth, D. G., in Methods in Enzymology (edited by C.-H. W. Hirs), Academic Press, New York 1967, Vol. XI, p Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L., Cooper, A., and Margoliash, E., J. Biol. Chem., in press. 24. Sokolovsky, M., Aviram, I., and Schejter, A., Biochemistry, 9 (1970) Shechter, E., Saludjian, P., Biopolymers, 8 (1967) Margoliash, E., Frohwirt, N., and Wiener, E., Biochem. J. 71 (1959) Schejter, A., and Aviram, I., J. Biol. Chem. 245 (1970) A. Schejter, T. Goldkorn, and M. Sokolovsky Department of Biochemistry, Tel-Aviv University Ramat-Aviv, Tel-Aviv, Israel