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A Rapid and Specific Method for Isolation of Thiol-Containing Peptides

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A Rapid and Specific Method for Isolation of Thiol-Containing Peptides Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3029-3033, 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-(gluta- protein 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 amino acid resi- KABI, Sweden. dues and one cysteine 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; iron (II).oxygen oxidoreduc- amounts of proteolytic fragments as determined by gel fil- tase] (ceruloplasmin) (1065 residues and three cysteines). 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 alkylation with radioactive iodoacetic acid 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 papain (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 (ethylene diamine tetraacetic acid) was included * Present address: Shemyakin Institute of Bioorganic Chemistry, to trap the Cu2+ ions of the enzyme. 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 25, 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:substrate 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 pyridine/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 glutathione 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.
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