Fragments of human growth hormone produced by digestion with thrombin: chemistry and biological properties.

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 220C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of fl-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCI and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCI. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1-135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143-191, 145-191 and 146-191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1-135 4peptide 143-191, peptide 1-135+peptide 145-191 and peptide 1-135+peptide 146-191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to he somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [14C]g]ueose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing USl-labeled human GH bound to antibodies to human GH.