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Isolation and Characterization of Fragments of Reduced and S-Carbamidomethylated Human Growth Hormone Produced by Plasmin Digestion. I. Chemistry^*

JOHN B. MILLS, JACK L. KOSTYO, MARTHA H. MOSELEY, CHARLES R. REAGAN and ALFRED E. WILHELMI

Departments of Biochemistry and Physiology, Emory University School of Medicine Atlanta, Georgia 30322

Abstract

Reduced and S-carbamidomethylated human GH (RCAM-hGH) was digested with human plasmin, yielding a mixture of products. These were partially separated by chromatography on DEAE-cellulose, yielding three major fractions: Da and Db, which were equipotent with native hGH in the weight gain test; and Dc, which was about half as active as hGH. Each of these was further purified by gel filtration, yielding a number of subtractions which were characterized as follows: Dal is a very stable noncovalent complex of residues 1–134 and 141–191 of RCAM-hGH; Da2 represents residues 20–41; Dbl is very similar to Dal, but appears to have lost one or more amide groups; Db3 represents residues 95–134; Dc2 is a heterogeneous fraction containing a further deamidated version of Dal and Dbl plus a similar complex of residues 42–134 and 141–191, apparently with some carboxyterminal heterogeneity; Dc3, like Db3, represents residues 95–134. The biological activities of these fragments are discussed in the accompanying paper.

Earlier work has shown that native hGH, upon digestion with plasmin, is cleaved primarily at residues 134 and 140. It is shown here that when RCAM-hGH is digested with plasmin, in about 87% of the molecules at least one cleavage takes place in addition to those at residues 134 and 140. (Endocrinology 102: 1366, 1978)

Footnotes

^* This work was supported by USPHS Grants AM 03598, HD 01231, and HD 08230 and The Kroc Foundation, Santa Ynez, California.

To whom requests for reprints should be addressed.

Received October 25, 1977.