The amino acid composition of the hemerythrin from the sipunculid worm Golfingia gouldii is, in residues per molecule of protein subunit of 13,500 molecular weight: Lysu.o,
Peptides from two separate tryptic digests of Golfingia gouldii hemerythrin were purified by Dowex 50W-X2 column chromatography followed by paper electrophoresis, paper chromatography, gel filtration, or various combinations of these techniques. The amino acid sequences of these peptides were completely determined, except for the relative positions of some of the residues in three peptides. These sequences account for a total of 113 residues T Xhe preceding paper (Groskopf et al., 1966) gives the amino acid composition as well as the aminoterminal and the carboxyl-terminal sequences of Golfingia gouldii hemerythrin. The present paper will describe the amino acid sequences of all the peptides recovered from tryptic digests of the protein. Experimental SectionPreparation of Tryptic Digests. G. gouldii hemerythrin was prepared and crystallized, and the ironfree protein was recovered, as previously described (Groskopf et al., 1966). Two separate tryptic digests, one from the whole protein and the other from the iron-free protein, were utilized.In the first case, 2.5 g (179 /¿moles of subunit) of oxyhemerythrin, dissolved in 435 ml of pH 8.0, 0.02 m ammonium bicarbonate, was denatured by heating at 65°for 5 min. The resulting suspension was cooled, and to it was added 25 mg of trypsin (Worthington Biochemical Corp.) treated with dilute hydrochloric acid according to Northrop and Kunitz (1936) to destroy residual chymotryptic activity. Further additions of 25 mg of trypsin were made at 4 and 8 hr and the hydrolysis was allowed to proceed at 32°for 22 hr. The digest was lyophilized. The peptides recovered from this digest are denoted T.
The mechanism of activation of human plasminogen by streptokinase has been shown to be due to the cleavage of a single arginyl-valine bond ( 1, 2). Others suggested that this activation mechanism may be an indirect one involving first the formation of an activator complex, an equimolar complex of human plasmin and streptokinase (3-5). The direct activation involving the cleavage of a peptide bond did not appear to be possible since a synthetic peptide substrate has not been found for streptokinase, whereas all other well-defined activators of human plasminogen, e.g., urokinase, trypsin, and pigheart activator, have the ability to cleave peptide bonds (6-8). To show whether the direct, indirect, or both mechanisms are operable, would require the removal or inhibition of the plasmin contaminant from the human plasminogen preparation by either physical or chemical methods.Previous work from our laboratory showed that streptokinase can activate a human plasminogen preparation in which all plasmin activity had been effectively inhibited by prior treatment with DFP (9). Experiments will be described which show that streptokinase can activate human plasminogen directly, rather than through a preexistent active equimolar human plasmin-s t r eptokinase complex.Materials and Methods. Human plasminogen was prepared from Fraction 1112,3 by previously described methods (10). Human plasmin was prepared by activating human plasminogen with either streptokinase or urokinase in 25% glycerol (2, 10). The specific activities of the plasminogen and plasmin preparations used in this study were 24-28 casein units/mg of protein (11). Bovine plasminogen was obtained from Parke-Davis and Company. The specific proteolytic Grant HE-04366.* Supported in part by U. S. Public Health Service activity was 0.4 casein units/mg of solids. A commercial preparation of partially purified streptokinase (Varidase, Lederle Laboratories) containing approximately 5000 units/ mg of solids was used.Human plasminogen was incubated in M diisopropyl phosphorofluoridate (DFP) (Aldrich Chemical Company) for 30 min at 0", at pH 9.0 (9). Under these conditions, the plasmin contaminant in the plasminogen preparation was completely inhibited (40-fold excess of enzyme assayed). An equimolar human plasmin-streptokinase complex ( 12) (bovine plasminogen activator) wits also incubated in M DFP as described above. Complete inhibition of both proteolytic and bovine plasminogen activator activities was observed ( 100-fold excess of enzyme assayed) (9). Proteolytic and bovine plasminogen activator activities were determined by procedures previously described (13).Horizontal starch gel electrophoresis was carried out in the Pherograph (Hormuth) in 8 M urea-0,017 M sodium formate0.05 M 2-mercaptoethanol, pH 3.2, as previously described ( 1). Electrophoresis of human plasminogen and plasmin was performed on cellulose acetate membranes at 50 V/cm at room temperature for 30-50 min using the Beckman Microzone system. Protein samples were dissolved in a 0.075 M sodium barbital-0.0...
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