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Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3029-3033, August 1975 A rapid and specific method for isolation of -containing from large by thiol- exchange on a solid support ( 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-(gluta- has been determined (5). Bovine serum albumin was thione-2-pyridyl disulfide) conjugate] has been used to immo- obtained from Sigma Chem. Co. (St. Louis, Mo.). It con- bilize proteins with a single or a few thiol groups via disul- tained 59% mercaptalbumin, the form of the fide bridges. The immobilized proteins were subsequently i.e., protein proteolytically degraded. After washing, the thiol-containing with a free thiol group. The thiol concentration was deter- peptides were eluted with a reducing agent. A single prepar- mined by the method of Grassetti and Murray (6). The ative paper electrophoresis, occasionally after a modification amino-acid sequence of the NH2-terminal part of the pro- such as oxidation, was sufficient to obtain pure peptides in tein has been determined (7). Human ceruloplasmin, pre- good yields. The method was applied to the major parval- pared from retroplacental serum, was obtained from AB bumin from hake muscle (a protein with 108 resi- KABI, Sweden. dues and one residue), to mercaptalbumin from bo- Stockholm, The protein had a A610/A280 vine serum (565 residues and one cysteine), and to human ratio indicating 95% purity and contained only small serum ferroxidase [EC 1.16.3.1; (II). oxidoreduc- amounts of proteolytic fragments as determined by gel fil- tase] (ceruloplasmin) (1065 residues and three ). The tration in 6 M guanidine-HCl (8). Its cysteine content, as de- use of the technique, e.g., as a simple means of obtaining ho- termined by with radioactive with mologous peptides in related proteins, is discussed. known specific activity (9), was 3 mol per mol of protein as- suming a molecular weight of 134,000. The advent of specific adsorbents containing activated thiol Chemicals. Guanidine-hydrochloride was grade I ob- groups has permitted the reversible covalent attachment of tained from Sigma Chem. Co. 2,2'-Dipyridyl disulfide was cysteinyl-proteins to a solid phase (1). The technique, which the product of Aldrich-Europe, Beerse, Belgium. Other has been called covalent chromatography, has been used for chemicals were of analytical preparation of pure and highly active (1), the isola- grade. tion of mercaptalbumin (2), and the reversible immobiliza- tion of urease (3). We now want to report how the same ad- sorbent can be used for the specific isolation of thiol-contain- Preparative methods ing peptides from proteolytic digests in essentially a single Covalent Attachment of Thiol Proteins. Activated thiol- step. Sepharose was washed with a large excess of coupling buffer The method has been applied to the major parvalbumin on a glass filter and then suspended in approximately a 5- from hake muscle, bovine serum mercaptalbumin, and fold volume of buffer. Protein (0.5-1.0 ,umol) was added to a human serum ferroxidase [EC 1.16.3.1; iron (II):oxygen oxi- 10-fold molar excess of active groups. Air was excluded by doreductase] (ceruloplasmin). The first of these is a small bubbling nitrogen through the suspension. The mixture was protein of known sequence and a single cysteinyl residue, rotated end over end at room temperature for an appropri- while the other two are both very large proteins with one ate time (see the individual experiments below). After cou- and three cysteinyl residues, respectively, in addition to a pling, the suspension was packed in a small chromatography number of disulfide bridges. column and the buffer collected by elution. The 2-thiopyridone that is formed in the reaction has an EXPERIMENTAL absorbance maximum at 343 nm (1), with a molar absorptiv- Materials ity of 7.06 X 103 (6). The coupling yield could, therefore, be Adsorbent. Activated thiol-Sepharose 4B [agarose-(gluta- determined from the absorbance at 343 nm of the eluate. thione-2-pyridyl disulfide)] was obtained from Pharmacia The absorbance at 280 nm of the nonadsorbed protein can Fine Chemicals AB, Uppsala, Sweden. The gel contained also be used to follow the reaction. In this case the contribu- about 40 microequivalents of active 2-pyridyl disulfide tion of the 2-thiopyridone at this wavelength, which is 1.25 structures per g of dry gel, as determined according to X Am (6), has to be subtracted. The thiol content of the sus- Brocklehurst et al. (1). After use the gel can be reactivated pension, determined with 2,2'-dipyridyl disulfide (6), offers by reaction with 2,2'-dipyridyl disulfide (1). a third possibility for following the coupling reaction. Proteins. The major parvalbumin from hake muscle was A buffer with a pH close to 8 was used for the coupling prepared as described by Pechere et al. (4) and used as a ly- (see Results). The buffers contained 6 M guanidine-HCI in ophilized powder. The complete amino some of the experiments. In the coupling of ceruloplasmin, 1 acid sequence of this mM EDTA ( diamine tetraacetic acid) was included * Present address: Shemyakin Institute of Bioorganic , to trap the Cu2+ of the . Academy of Sciences of the USSR, Moscow, USSR. Digestion of Immobilized Proteins. The column was tTo whom reprint requests should be sent. equilibrated with one total volume of the buffer to be used 3029 Downloaded by guest on September 29, 2021 3030 Biochemistry: Egorov et al. Proc. Nat. Acad. Sci. USA 72 (1975)

in the proteolytic digestion and transferred to a plastic bottle Table 1. Immobilization of proteins to thiol-Sepharose with several volumes of buffer. For the peptic digestions this was formic acid/acetic acid/water (1:4:45, v/v/v) (10). Pep- Cou- sin (Worthington) dissolved in water was added to give a pling final enzyme: ratio of 1:25 (w/w). The bottle was Time yield Coupling buffer (hr) Protein (%) then rotated end over end in a 370 water bath over night (15 hr). 0.1 M Tris*HCl pH 8.0, Detachment of Cysteinylpeptides. After digestion the 6 M guanidine-HCl 2 Parvalbumin 94 gel was again packed in a chromatography column. The first 0.1 M sodium phosphate eluate, containing the nonattached peptides, was collected pH 7.8 6 Bovine serum and the gel was washed with several total volumes of 0.2 M mercaptalbumin 97 ammonium acetate, pH 8.6. A wash with 1 M NaCl was in- 0.2 M N-Ethylmorpholine cluded to elute nonspecifically adsorbed peptides. Now one acetate pH 8.0, 6 M total volume of 20 mM 2-mercaptoethanol in 0.2 M ammo- guanidine-HCl, nium acetate, pH 8.6, was introduced, and the column was 1 mM EDTA 15 Ceruloplasmin 20-30 left for 30 min at 250 to allow complete reaction. The elu- tion was then continued with the reducing buffer, and the All experiments were performed at 250. All other conditions were as stated. Recovery was estimated from absorbance at 343 nm of the desorbed peptides were collected. 2-thiopyridone liberated in the reaction, after elution of the buffer Preparative Paper Electrophoresis. The peptide frac- in a small chromatographic column, except for ceruloplasmin, tions obtained were lyophilized to reduce the volume and where the yield was estimated from the recovery of the pure pep- evaporate buffer salts and 2-mercaptoethanol. Preparative tides (see text). paper electrophoresis was performed using Whatman 3 MM values. The reaction on the gel attains practical rates above paper and a Gilson Medical Electronics high-voltage paper pH 7. Buffers around pH 8, such as sodium phosphate and electrophoresis equipment or (at pH 1.9) a small low-voltage ammonium acetate, were used routinely. Attachment is, (440 V) paper electrophoresis apparatus. The buffers used however, possible also close to pH 2, used for peptic diges- were 1.24 M /0.069 M acetic acid (pH 6.46), 0.041 tion, even if the coupling reaction is slower in the acidic pH M pyridine/0.58 M acetic acid (pH 3.50), and 1.37 M acetic range. acid/0.66 M formic acid (pH 1.9). Guide strips were stained with a 0.2% solution of ninhydrin in ethanol/acetic acid/col- Immobilization and digestion of proteins lidine (60:20:8, v/v/v). Peptides were eluted from the paper Conditions for and yields of immobilization of the proteins with either 10% acetic acid or water. to the activated thiol-Sepharose are summarized in Table 1. Analytical methods The reaction proceeded with good yields in the case of par- valbumin and serum albumin. In spite of a considerably Paper Electrophoresis. The peptides prepared were all longer coupling time, the yield for ceruloplasmin was rather analyzed by paper electrophoresis on Whatman 1 paper. poor. This is probably explained by the presence of three The buffers and equipment used were the same as described thiol groups on a single peptide chain. When the first group above for the preparative procedures, as was also the stain- has reacted, the two following might react slower with acti- ing technique. An amino-acid calibration mixture as well as vated structures because of the excluded volume effect in a sample of were used as references. the guanidine.HCl solution. For ceruloplasmin it was not Amino-Acid Analysis. Samples were hydrolyzed and ana- possible to evaluate the coupling yield from the spectrum lyzed on a Durrum D-500 amino-acid analyzer as described since a large background was present. In this case the cou- by Ryden and Eaker (11). pling yield was instead estimated from the final yield of the peptides. RESULTS AND DISCUSSION A further complication arises from the possibility that a disulfide bridge might be located so close to a cysteine resi- General approach due that they will be contained in the same peptide after di- The aim of the present work was to attach cysteinyl residues gestion. In this case one peptide with two or more cysteine of proteins to the activated thiol gel and thereby specifically residues and one cysteine peptide derived from the disulfide isolate the peptides of which they were a part. This could be bridge will be obtained after elution of the column after re- achieved in several different ways, in particular: (i) diges- duction. tion of the protein and attachment of cysteinyl peptides or In the experiments described pepsin was used throughout (ii) attachment of protein to the activated gel and digestion as the proteolytic enzyme. It is also possible to use other en- of the immobilized protein. Both these approaches were pos- zymes, as shown by preliminary results. The relatively good sible, as shown in preliminary experiments. We have chosen recovery of peptides (see below) indicates that the proteins to start by attaching the protein to the activated gel, since are readily available for digestion when immobilized on the then no thiol groups were available for oxidation during the gel. A large amount of enzyme can be used since it is washed digestion. out from the gel after the digestion. In several proteins the -SH groups are not accessible to the . The major parvalbumin from hake muscle and Preparation of cysteine-containing peptides human ceruloplasmin used here both belong to this cate- The Major Parvalbumin from Hake Muscle. Analytical gory. In these cases an unfolding agent was added to the electrophoresis of the two eluates from the digestion of im- coupling mixture. Both and guanidine-HCl proved pos- mobilized parvalbumin is shown in Fig. 1. The fraction elut- sible to use, but since guanidine.HCl in general is more effi- ed with the reducing agent contained one major component cient, it was used routinely. and two contaminants. Amino acid analysis of this fraction The immobilization could be carried out at different pH directly did not give a clean result. A preparative electro- Downloaded by guest on September 29, 2021 Biochemistry: Egorov et al. Proc, Nat. Acad. Sci. USA 72 (1975) 3031 c athode

Cp-l Pa-2

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 pro- teins immobilized on thiol-Sepharose. Electrophoresis was conducted for 25 min at 4000 V. Cathode upwards. (a) Peptides from hake parval- bumin eluted without reduction. (b) Peptides from hake parvalbumin (Pa = parvalbumin peptide) eluted with reducing agent (GSH = glu- tathione). (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 refer- ence. (g) Peptides from bovine serum albumin eluted without reduction. (h) Peptides from bovine serum albumin (BSA = bovine serum al- bumin peptide) eluted with reducing agent. (i) Amino-acid reference mixture (same as e). phoresis at pH 6.46 was therefore conducted, and all the single major component. The major trace contaminant was peptides were recovered and analyzed. The composition of identified as glutathione by mobility. In this case the gel had the main peptide (Pa-i) (Table 2) proved this to consist of been treated with reducing agents and reactivated with residues 16 through 29 in the protein, with about 14% of the 2,2'-dipyridyl disulfide before immobilization, as described peptide also having residues 14-15 (Ala-Leu) left. This is the above, to avoid glutathione leakage. This was successful only peptide that has been obtained earlier from the peptic digest to a limited degree. The amount eluted was estimated to be of free parvalbumin (5). Peptide Pa-2 was clearly related to 0.1% of the amount on the gel. The cysteine-peptide was Pa-i, but was not completely pure. The yield of peptide further purified by preparative paper electrophoresis at pH Pa-i was 40% and of Pa-2 2% (together, 42%). The recov- 3.50. Analysis of the peptide (see Table 2) shows it to be an eries in paper electrophoresis are expected to be 30-80%. octapeptide identical in composition with the thiolpeptide The total recovery of peptide thus indicates that the diges- isolated by Witter and Tuppy (12) from a peptic digest of tion of immobilized protein had proceeded reasonably well. the protein except for the difference of one The major contaminant was shown by composition to consist residue. The difference is explained by the revision of this of glutathione. The yield corresponded to a 1% leakage of part of the sequence by King and Spencer (7), where the glutathione from the gel. It is likely that the glutathione ob- peptide constitutes residues 31-38 in the albumin peptide tained in this way is attached to the gel via a disulfide bridge chain. It therefore seems that pepsin hydrolyzes the same and is, therefore, eluted by the reducing agent in the desorp- bonds in the immobilized and the free form of bovine serum tion. albumin. The recovery of the peptide (77%) indicated that Bovine Serum Albumin. Albumin consists of a single pep- the peptic digestion was essentially quantitative, assuming tide chain of about 565 amino-acid residues and a single cys- some loss in the elution from the paper in the electrophoresis teinyl residue. It was attached to the gel without having de- step. naturants present. The eluate after peptic digestion, as ana- Ceruloplasmin. This protein consists of a single peptide lyzed by paper electrophoresis (Fig. 1), here too contained a chain of about 1065 residues and it has three cysteinyl resi- Downloaded by guest on September 29, 2021 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) 0.99 (1) 2.02 (2) 1.17 (1) 4.80 (5) 0.54 (1) 1.05 (1) 1.65 (2) 1.14 (1) 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) 1.04 (1) 0.89 (1) 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 0.14 0.88 (1) 3.22 (3) 1.84 (2) 0.98 (1) Phenylalanine 1.95 (2) 0.89 (1) 1.00 (1) 1.06 (1) 3.96 (4) 2.00 (2) 0.73 (1) 2.70 (3) 14 8 13 16 19 Recovery (%) 40 77 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 pep- and often functionally important. In these instances it might tides from the protein proved to be difficult because of the be of interest to isolate and study selectively the portions of a complexity of the peptide mixtures obtained. The new ap- big protein that contain these groups. Ceruloplasmin is an proach was thus tried. The electrophoretic analysis of the example of a protein whose sequence is not determined as a thiol peptides is shown in Fig. 1. Five major components whole without a considerable effort. With the new tech- were resolved at pH 6.46. These were fractionated by suc- nique the few cysteine-containing parts of the protein can, cessive preparative electrophoretic runs at pH 6.46 and 1.90, however, be isolated easily and studied with regard to se- the second one after performic acid oxidation. Seven cys- quence homologies or binding. Similarly, disulfide teinyl peptides were isolated in this way. The composition of bridges are repeatedly highly conserved in evolution..These peptides Cp-lb, Cp-2, and Cp-4 are shown in Table 2. can also be isolated by the described method after alkylation (Lower case letters refer to the separation obtained at pH of the cysteinyl thiol groups and subsequent reduction. The 1.90.) Peptide Cp-la was identical to Cp-2, except for the possibility of introducing thiol groups on other amino acid loss of one glutamic acid. Peptides Cp-3a and Cp-3b were side chains (16) further extends the possible applications of both rather pure and related to Cp-lb. The recovery of sev- the method. eral different peptides from a single cysteinyl sequence is Specific reversible covalent attachment to a solid phase as consistent with the low specificity of pepsin. Finally the a means for isolating peptides has several advantages over most anodic peptide was glutathione. The yields were rather earlier methods. It is extremely simple to carry out, and it is poor which was expected after the several steps that were not dependent upon separations on column or paper with performed. Nevertheless, these peptides were obtained after concomitant analysis and, sometimes, modification reac- a few days of work in striking contrast to the earlier unsuc- tions. It should indeed be possible to extend the solid phase cessful trials. approach to several of the existing specific and reversible modification reactions of amino acid side chains if these re- Comments on the method actions are used for designing new appropriate absorbents. Previous available methods for the specific purification of cysteine-containing peptides all rely on a diagonal tech- We thank Miss H. Norder for doing some of the preparative runs nique, i.e., a specific modification of the thiol group before on the ceruloplasmin peptides and Dr. D. Eaker for amino acid the repeat of a purification step either on paper as first sug- analysis and discussions. The project has been supported by grants The Swedish Natural Science Research Council Brown and or on for from (K-3568-001 gested by Hartley (13) column example and K-3477-003), The Swedish Academy of Engineering Science as suggested by Franek and Novotny (14). In the approach from The Foundation to the Memory of Bengt Lundqvist, and from described here this two-step procedure is replaced by a sin- the Lennander Foundation. gle step, the specific covalent attachment of the thiol group to a solid phase support. A similar approach was reported by 1. Brocklehurst, K., Carlsson, J., Kierstan, M. P. J. & Crook, E. der Terrossian et. al. (15), who separated the non-identical M. (1973) Biochem. J. 133,573-584. subunits of lombricine kinase from Lumbricus terrestris 2. Carlsson, J. & Svenson, A. (1974) FEBS Lett. 42, 183-186. muscle by chromatography on a Sepharose-mercurial gel. 3. Carlsson, J., Axen, R., Brocklehurst, K. & Crook, E. M. (1974) Cysteinyl residues in proteins are comparatively scarce Eur. J. Biochem. 44,189-194. Downloaded by guest on September 29, 2021 Biochemistry: Egorov et al. Proc. Nat. Acad. Sci. USA 72 (1975) 303

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