from large proteins by thiol-disulfide exchange on a solid support (peptide separation/cysteinyl peptides/covalent chromatography)

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1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp , August 1975 Biochemistry A rapid and specific method for isolation of thiol-containing peptides from large proteins by thiol-disulfide exchange on a solid support (peptide separation/cysteinyl peptides/covalent chromatography) T. A. EGOROV*, ANDERS SVENSON, LARS RYDtN, AND JAN CARLSSONt Institute of Biochemistry, University of Uppsala, Uppsala, Sweden Communicated by H. A. Scheraga, May 27, 1975 ABSTRACT Activated thiol-sepharose [agarose-(glutathione-2-pyridyl disulfide) conjugate] has been used to immobilize proteins with a single or a few thiol groups via disulfide bridges. The immobilized proteins were subsequently proteolytically degraded. After washing, the thiol-containing peptides were eluted with a reducing agent. A single preparative paper electrophoresis, occasionally after a modification such as oxidation, was sufficient to obtain pure peptides in good yields. The method was applied to the major parvalbumin from hake muscle (a protein with 108 amino acid residues and one cysteine residue), to mercaptalbumin from bovine serum (565 residues and one cysteine), and to human serum ferroxidase [EC ; iron (II).oxygen oxidoreductase] (ceruloplasmin) (1065 residues and three cysteines). The use of the technique, e.g., as a simple means of obtaining homologous peptides in related proteins, is discussed. The advent of specific adsorbents containing activated thiol groups has permitted the reversible covalent attachment of cysteinyl-proteins to a solid phase (1). The technique, which has been called covalent chromatography, has been used for preparation of pure and highly active papain (1), the isolation of mercaptalbumin (2), and the reversible immobilization of urease (3). We now want to report how the same adsorbent can be used for the specific isolation of thiol-containing peptides from proteolytic digests in essentially a single step. The method has been applied to the major parvalbumin from hake muscle, bovine serum mercaptalbumin, and human serum ferroxidase [EC ; iron (II):oxygen oxidoreductase] (ceruloplasmin). The first of these is a small protein of known sequence and a single cysteinyl residue, while the other two are both very large proteins with one and three cysteinyl residues, respectively, in addition to a number of disulfide bridges. EXPERIMENTAL Materials Adsorbent. Activated thiol-sepharose 4B [agarose-(glutathione-2-pyridyl disulfide)] was obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. The gel contained about 40 microequivalents of active 2-pyridyl disulfide structures per g of dry gel, as determined according to Brocklehurst et al. (1). After use the gel can be reactivated by reaction with 2,2'-dipyridyl disulfide (1). Proteins. The major parvalbumin from hake muscle was prepared as described by Pechere et al. (4) and used as a lyophilized powder. The complete amino acid sequence of this * Present address: Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR, Moscow, USSR. tto whom reprint requests should be sent protein has been determined (5). Bovine serum albumin was obtained from Sigma Chem. Co. (St. Louis, Mo.). It contained 59% mercaptalbumin, i.e., the form of the protein with a free thiol group. The thiol concentration was determined by the method of Grassetti and Murray (6). The amino-acid sequence of the NH2-terminal part of the protein has been determined (7). Human ceruloplasmin, prepared from retroplacental serum, was obtained from AB KABI, Stockholm, Sweden. The protein had a A610/A280 ratio indicating 95% purity and contained only small amounts of proteolytic fragments as determined by gel filtration in 6 M guanidine-hcl (8). Its cysteine content, as determined by alkylation with radioactive iodoacetic acid with known specific activity (9), was 3 mol per mol of protein assuming a molecular weight of 134,000. Chemicals. Guanidine-hydrochloride was grade I obtained from Sigma Chem. Co. 2,2'-Dipyridyl disulfide was the product of Aldrich-Europe, Beerse, Belgium. Other chemicals were of analytical grade. Preparative methods Covalent Attachment of Thiol Proteins. Activated thiol- Sepharose was washed with a large excess of coupling buffer on a glass filter and then suspended in approximately a 5- fold volume of buffer. Protein ( ,umol) was added to a 10-fold molar excess of active groups. Air was excluded by bubbling nitrogen through the suspension. The mixture was rotated end over end at room temperature for an appropriate time (see the individual experiments below). After coupling, the suspension was packed in a small chromatography column and the buffer collected by elution. The 2-thiopyridone that is formed in the reaction has an absorbance maximum at 343 nm (1), with a molar absorptivity of 7.06 X 103 (6). The coupling yield could, therefore, be determined from the absorbance at 343 nm of the eluate. The absorbance at 280 nm of the nonadsorbed protein can also be used to follow the reaction. In this case the contribution of the 2-thiopyridone at this wavelength, which is 1.25 X Am (6), has to be subtracted. The thiol content of the suspension, determined with 2,2'-dipyridyl disulfide (6), offers a third possibility for following the coupling reaction. A buffer with a ph close to 8 was used for the coupling (see Results). The buffers contained 6 M guanidine-hci in some of the experiments. In the coupling of ceruloplasmin, 1 mm EDTA (ethylene diamine tetraacetic acid) was included to trap the Cu2+ ions of the enzyme. Digestion of Immobilized Proteins. The column was equilibrated with one total volume of the buffer to be used

2 3030 Biochemistry: Egorov et al. in the proteolytic digestion and transferred to a plastic bottle with several volumes of buffer. For the peptic digestions this was formic acid/acetic acid/water (1:4:45, v/v/v) (10). Pepsin (Worthington) dissolved in water was added to give a final enzyme:substrate ratio of 1:25 (w/w). The bottle was then rotated end over end in a 370 water bath over night (15 hr) Ḋetachment of Cysteinylpeptides. After digestion the gel was again packed in a chromatography column. The first eluate, containing the nonattached peptides, was collected and the gel was washed with several total volumes of 0.2 M ammonium acetate, ph 8.6. A wash with 1 M NaCl was included to elute nonspecifically adsorbed peptides. Now one total volume of 20 mm 2-mercaptoethanol in 0.2 M ammonium acetate, ph 8.6, was introduced, and the column was left for 30 min at 250 to allow complete reaction. The elution was then continued with the reducing buffer, and the desorbed peptides were collected. Preparative Paper Electrophoresis. The peptide fractions obtained were lyophilized to reduce the volume and evaporate buffer salts and 2-mercaptoethanol. Preparative paper electrophoresis was performed using Whatman 3 MM paper and a Gilson Medical Electronics high-voltage paper electrophoresis equipment or (at ph 1.9) a small low-voltage (440 V) paper electrophoresis apparatus. The buffers used were 1.24 M pyridine/0.069 M acetic acid (ph 6.46), M pyridine/0.58 M acetic acid (ph 3.50), and 1.37 M acetic acid/0.66 M formic acid (ph 1.9). Guide strips were stained with a 0.2% solution of ninhydrin in ethanol/acetic acid/collidine (60:20:8, v/v/v). Peptides were eluted from the paper with either 10% acetic acid or water. Analytical methods Paper Electrophoresis. The peptides prepared were all analyzed by paper electrophoresis on Whatman 1 paper. The buffers and equipment used were the same as described above for the preparative procedures, as was also the staining technique. An amino-acid calibration mixture as well as a sample of glutathione were used as references. Amino-Acid Analysis. Samples were hydrolyzed and analyzed on a Durrum D-500 amino-acid analyzer as described by Ryden and Eaker (11). RESULTS AND DISCUSSION General approach The aim of the present work was to attach cysteinyl residues of proteins to the activated thiol gel and thereby specifically isolate the peptides of which they were a part. This could be achieved in several different ways, in particular: (i) digestion of the protein and attachment of cysteinyl peptides or (ii) attachment of protein to the activated gel and digestion of the immobilized protein. Both these approaches were possible, as shown in preliminary experiments. We have chosen to start by attaching the protein to the activated gel, since then no thiol groups were available for oxidation during the digestion. In several proteins the -SH groups are not accessible to the solvent. The major parvalbumin from hake muscle and human ceruloplasmin used here both belong to this category. In these cases an unfolding agent was added to the coupling mixture. Both urea and guanidine-hcl proved possible to use, but since guanidine.hcl in general is more efficient, it was used routinely. The immobilization could be carried out at different ph Table 1. Proc. Nat. Acad. Sci. USA 72 (1975) Immobilization of proteins to thiol-sepharose Coupling Coupling buffer Time (hr) Protein yield (%) 0.1 M Tris*HCl ph 8.0, 6 M guanidine-hcl 2 Parvalbumin M sodium phosphate ph Bovine serum mercaptalbumin M N-Ethylmorpholine acetate ph 8.0, 6 M guanidine-hcl, 1 mm EDTA 15 Ceruloplasmin All experiments were performed at 250. All other conditions were as stated. Recovery was estimated from absorbance at 343 nm of the 2-thiopyridone liberated in the reaction, after elution of the buffer in a small chromatographic column, except for ceruloplasmin, where the yield was estimated from the recovery of the pure peptides (see text). values. The reaction on the gel attains practical rates above ph 7. Buffers around ph 8, such as sodium phosphate and ammonium acetate, were used routinely. Attachment is, however, possible also close to ph 2, used for peptic digestion, even if the coupling reaction is slower in the acidic ph range. Immobilization and digestion of proteins Conditions for and yields of immobilization of the proteins to the activated thiol-sepharose are summarized in Table 1. The reaction proceeded with good yields in the case of parvalbumin and serum albumin. In spite of a considerably longer coupling time, the yield for ceruloplasmin was rather poor. This is probably explained by the presence of three thiol groups on a single peptide chain. When the first group has reacted, the two following might react slower with activated structures because of the excluded volume effect in the guanidine.hcl solution. For ceruloplasmin it was not possible to evaluate the coupling yield from the spectrum since a large background was present. In this case the coupling yield was instead estimated from the final yield of the peptides. A further complication arises from the possibility that a disulfide bridge might be located so close to a cysteine residue that they will be contained in the same peptide after digestion. In this case one peptide with two or more cysteine residues and one cysteine peptide derived from the disulfide bridge will be obtained after elution of the column after reduction. In the experiments described pepsin was used throughout as the proteolytic enzyme. It is also possible to use other enzymes, as shown by preliminary results. The relatively good recovery of peptides (see below) indicates that the proteins are readily available for digestion when immobilized on the gel. A large amount of enzyme can be used since it is washed out from the gel after the digestion. Preparation of cysteine-containing peptides The Major Parvalbumin from Hake Muscle. Analytical electrophoresis of the two eluates from the digestion of immobilized parvalbumin is shown in Fig. 1. The fraction eluted with the reducing agent contained one major component and two contaminants. Amino acid analysis of this fraction directly did not give a clean result. A preparative electro-

3 Biochemistry: Egorov et al. c athode Proc, Nat. Acad. Sci. USA 72 (1975) 3031 Pa-2 Cp-l Cp-2 c Cp-3... i,. #...- BSA-1 _ U, 4-: Cp-4 9; 'p GSH GSH GSH w a b c d e f g h i FIG. 1. Analytical high-voltage paper electrophoresis at ph 6.46 (a-e) and ph 3.50 (f-i) of peptides prepared by peptic digestion of proteins immobilized on thiol-sepharose. Electrophoresis was conducted for 25 min at 4000 V. Cathode upwards. (a) Peptides from hake parvalbumin eluted without reduction. (b) Peptides from hake parvalbumin (Pa = parvalbumin peptide) eluted with reducing agent (GSH = glutathione). (c) Peptides from human ceruloplasmin eluted without reduction. (d) Peptides from human ceruloplasmin (Cp = ceruloplasmin peptide) eluted with reducing agent. (e) Amino-acid reference mixture (Lys, His, neutral amino acids, Glu, and Asp). (f) Glutathione reference. (g) Peptides from bovine serum albumin eluted without reduction. (h) Peptides from bovine serum albumin (BSA = bovine serum albumin peptide) eluted with reducing agent. (i) Amino-acid reference mixture (same as e). phoresis at ph 6.46 was therefore conducted, and all the peptides were recovered and analyzed. The composition of the main peptide (Pa-i) (Table 2) proved this to consist of residues 16 through 29 in the protein, with about 14% of the peptide also having residues (Ala-Leu) left. This is the peptide that has been obtained earlier from the peptic digest of free parvalbumin (5). Peptide Pa-2 was clearly related to Pa-i, but was not completely pure. The yield of peptide Pa-i was 40% and of Pa-2 2% (together, 42%). The recoveries in paper electrophoresis are expected to be 30-80%. The total recovery of peptide thus indicates that the digestion of immobilized protein had proceeded reasonably well. The major contaminant was shown by composition to consist of glutathione. The yield corresponded to a 1% leakage of glutathione from the gel. It is likely that the glutathione obtained in this way is attached to the gel via a disulfide bridge and is, therefore, eluted by the reducing agent in the desorption. Bovine Serum Albumin. Albumin consists of a single peptide chain of about 565 amino-acid residues and a single cysteinyl residue. It was attached to the gel without having denaturants present. The eluate after peptic digestion, as analyzed by paper electrophoresis (Fig. 1), here too contained a single major component. The major trace contaminant was identified as glutathione by mobility. In this case the gel had been treated with reducing agents and reactivated with 2,2'-dipyridyl disulfide before immobilization, as described above, to avoid glutathione leakage. This was successful only to a limited degree. The amount eluted was estimated to be 0.1% of the amount on the gel. The cysteine-peptide was further purified by preparative paper electrophoresis at ph Analysis of the peptide (see Table 2) shows it to be an octapeptide identical in composition with the thiolpeptide isolated by Witter and Tuppy (12) from a peptic digest of the protein except for the difference of one glutamic acid residue. The difference is explained by the revision of this part of the sequence by King and Spencer (7), where the peptide constitutes residues in the albumin peptide chain. It therefore seems that pepsin hydrolyzes the same bonds in the immobilized and the free form of bovine serum albumin. The recovery of the peptide (77%) indicated that the peptic digestion was essentially quantitative, assuming some loss in the elution from the paper in the electrophoresis step. Ceruloplasmin. This protein consists of a single peptide chain of about 1065 residues and it has three cysteinyl resi-

4 3032 Biochemistry: Egorov et al. Proc. Nat. Acad. Sci. USA 72 (1975) Table 2. Amino-acid composition of cysteine-containing peptides purified by paper electrophoresis after elution from thiol-sepharose Mercapt- Ceruloplasmin Parvalbumin albumin Amino acid Pa-1 BSA-1 Cp-lb Cp-2 Cp-4 Cysteicacid 1.01 (1) 1.08 (1) 0.76(1) 0.83 (1) 0.82(1) Aspartic acid 0.99 (1) 2.02 (2) 1.17 (1) 4.80 (5) Methionine sulfone 0.54 (1) 1.05 (1) Threonine 1.65 (2) 1.14 (1) Serine 1.00(1) 1.11 (1) 1.49(1) Glutamic acid 2.07 (2) 3.06 (3) 1.33 (1) 1.32 (1) 3.57 (3) Proline 1.04 (1) 0.89 (1) Glycine 2.07 (2) 1.35 (1) 1.40 (1) 0.81 (1) Alanine 3.18 (3) 1.16(1) 1.18 (1) Valine 1.00 (1) 1.06 (1) Isoleucine 1.02 (1) 1.05 (1) Leucine (1) 3.22 (3) 1.84 (2) Tyrosine 0.98 (1) Phenylalanine 1.95 (2) 0.89 (1) Histidine 1.00 (1) 1.06 (1) 3.96 (4) Lysine 2.00 (2) 0.73 (1) 2.70 (3) Arginine Recovery (%) Cysteine was determined as cysteic acid after performic acid oxidation. The values are expressed as mol/mol of peptide. Abbreviations: Pa = parvalbumin, BSA = bovine serum mercaptalbumin, and Cp = ceruloplasmin. dues. Previous attempts to isolate cysteine-contalning peptides from the protein proved to be difficult because of the complexity of the peptide mixtures obtained. The new approach was thus tried. The electrophoretic analysis of the thiol peptides is shown in Fig. 1. Five major components were resolved at ph These were fractionated by successive preparative electrophoretic runs at ph 6.46 and 1.90, the second one after performic acid oxidation. Seven cysteinyl peptides were isolated in this way. The composition of peptides Cp-lb, Cp-2, and Cp-4 are shown in Table 2. (Lower case letters refer to the separation obtained at ph 1.90.) Peptide Cp-la was identical to Cp-2, except for the loss of one glutamic acid. Peptides Cp-3a and Cp-3b were both rather pure and related to Cp-lb. The recovery of several different peptides from a single cysteinyl sequence is consistent with the low specificity of pepsin. Finally the most anodic peptide was glutathione. The yields were rather poor which was expected after the several steps that were performed. Nevertheless, these peptides were obtained after a few days of work in striking contrast to the earlier unsuccessful trials. Comments on the method Previous available methods for the specific purification of cysteine-containing peptides all rely on a diagonal technique, i.e., a specific modification of the thiol group before the repeat of a purification step either on paper as first suggested by Brown and Hartley (13) or on column for example as suggested by Franek and Novotny (14). In the approach described here this two-step procedure is replaced by a single step, the specific covalent attachment of the thiol group to a solid phase support. A similar approach was reported by der Terrossian et. al. (15), who separated the non-identical subunits of lombricine kinase from Lumbricus terrestris muscle by chromatography on a Sepharose-mercurial gel. Cysteinyl residues in proteins are comparatively scarce and often functionally important. In these instances it might be of interest to isolate and study selectively the portions of a big protein that contain these groups. Ceruloplasmin is an example of a protein whose sequence is not determined as a whole without a considerable effort. With the new technique the few cysteine-containing parts of the protein can, however, be isolated easily and studied with regard to sequence homologies or copper binding. Similarly, disulfide bridges are repeatedly highly conserved in evolution..these can also be isolated by the described method after alkylation of the cysteinyl thiol groups and subsequent reduction. The possibility of introducing thiol groups on other amino acid side chains (16) further extends the possible applications of the method. Specific reversible covalent attachment to a solid phase as a means for isolating peptides has several advantages over earlier methods. It is extremely simple to carry out, and it is not dependent upon separations on column or paper with concomitant analysis and, sometimes, modification reactions. It should indeed be possible to extend the solid phase approach to several of the existing specific and reversible modification reactions of amino acid side chains if these reactions are used for designing new appropriate absorbents. We thank Miss H. Norder for doing some of the preparative runs on the ceruloplasmin peptides and Dr. D. Eaker for amino acid analysis and discussions. The project has been supported by grants from The Swedish Natural Science Research Council (K and K ), The Swedish Academy of Engineering Science from The Foundation to the Memory of Bengt Lundqvist, and from the Lennander Foundation. 1. Brocklehurst, K., Carlsson, J., Kierstan, M. P. J. & Crook, E. M. (1973) Biochem. J. 133, Carlsson, J. & Svenson, A. (1974) FEBS Lett. 42, Carlsson, J., Axen, R., Brocklehurst, K. & Crook, E. M. (1974) Eur. J. Biochem. 44,

5 Biochemistry: Egorov et al. 4. Pechere J.-F., Capony, J.-P. & Ryden, L. (1971) Eur. J. Biochem. 23, Capony, J.-P., Ryden, L. D le, J. & Pechere J.-F. (1973) Eur. J. Biochem. 32, Grassetti, D. R. & Murray, J. F. (1967) Arch. Biochem. Riophys. 119, King, T. P. & Spencer, E. M. (1972) Arch. Biochem. Biophys. 153, Ryden, L. (1971) FEBS Lett. 18, Ryden, L. & Eaker, D. (1975) FEBS Lett.53, Proc. Nat. Acad. Sci. USA 72 (1975) Ambler, R. P. (1963) Biochem. J. 89, Ryden, L. & Eaker, D. (1974) Eur. J. Biochem. 44, Witter, A. & Tuppy, H. (1960) Biochim. Biophys. Acta, 45, Brown, J. R. & Hartley, B. S. (1966) Biochem. J. 101, Franek, F. & Novotny, J. (1973) FEBS Lett. 37, der Terrossian, E., Pradel, L. A., Kassab, R. & Desvages, G. (1974) Eur. J. Biochem. 45, Perham, N. R. & Thomas, J. 0. (1971) J. Mol. Biol. 62,

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Focus concept Purification of a novel seed storage protein allows sequence analysis and determination of the protein