A sulfur-containing compound found in acid hydrolysates of proteins was identified 30 years ago as a trisulfide: bis-(2-amino-2-carboxyethyl) trisulfide (cysteine2S3). At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine2S3 also existed in biological systems. Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of delta-aminolevulinate synthetase. Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182-Cys189 in human growth hormone. We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys53-Cys165. The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification. A tandem mass spectral analysis on a Sciex electrospray instrument localized an addition of 32 Da to the Cys53-Cys165 fragment. Elemental composition was determined by accurate mass measurement obtained by peak matching to a synthetic peptide and established that an extra sulfur atom was involved.

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Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two‐chain nature by tryptic mapping, N‐ and C‐terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr‐142 and Tyr‐143, in contrast to the two‐chain variant isolated from human pituitary glands, which has a clip after residue Phe‐139. The recombinant two‐chain form was further characterized by reversed‐phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate‐containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two‐chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one‐chain rhGH was demonstrated with an enzyme‐linked immunosorbent assay.

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