High Resolution High Performance Liquid Chromatography Fingerprinting of Purified Human Chorionic Gonadotropin Demonstrates that Oxidation Is a Cause of Hormone Heterogeneity*

Abstract

To characterize the structures of the various forms of hCG and its immunologically related fragments measured in blood, urine, and tissue culture media, we developed a highly sensitive HPLC tryptic fingerprinting system that can characterize as little as 15 micrograms of material. Standard preparations of the subunits of hCG purified from pregnancy urine were found to exhibit heterogeneity on reverse phase HPLC chromatography, complicating efforts to characterize such forms of hCG. The same type of chromatographic heterogeneity was observed when the hormone was reduced and S-carboxymethylated (RCM) and its RCM subunits separated on HPLC. One major and one minor peak were observed in the alpha- and beta-subunits from both native and RCM hormone, with a ratio of between 5:1 and 10:1. Development and application of a high resolution, semimicrobore HPLC fingerprint technique used together with an examination of selected peptides by amino acid sequencing and mass spectrometry defined the differences between these forms as oxidation. Using each of the isolated RCM subunit's major and minor HPLC peaks, the oxidation was shown to be reversible and probably due to interconversion of methionine with its sulfoxide form.

The Heterogeneity of Human Chorionic Gonadotropin (hCG). II. Characteristics and Origins of Nicks in hCG Reference Standards*

“…The first peak to elute, as shown by sequence analysis and SDS-gel analysis (23.6 min), was nicked hCG0. The next peak, eluting at 24.7-24.8 min, was a mixture of a-and 0-chains and was at the position identified in an earlier study (17) as that of a-subunit containing methionines oxidized to sulfoxides and 0-subunit with its single methionine likewise oxidized. The next two major peaks were the intact a-subunit (25.3 min) and the intact 0-subunit (26.1 min), as reported by Sakakibara et al (9).…”

“…Assays were performed using B109 antibody and [ 125 I] hCG as tracer, with both hCG CR127 and 100% nicked hCG preparation C5(10) as competitors, and were repeated twice. The procedures for SDS-gel electrophoresis and staining of gels were also described previously (17).…”

Molecular heterogeneity of the β-core fragment of human chorionic gonadotrophin

“…Quantitative differences in carbo¬ hydrate content probably account for most of the observed heterogeneity. More recently it was also shown that oxidation of specific methionine residues may also be responsible (Pollak et al 1990). …”

Facile separation of the subunits of ovine and bovine gonadotrophins by high-performance liquid chromatrography: microheterogeneity and biological activity

“…That these compo¬ nents were inherent in the preparation analysed and possibly in the tissue itself or during its collection and storage is suggested by the lack of interconversion from one to the other by laboratory manipulations of the purified oLH by such treatments as incubation in acid, alkali or neutral buffers or repurification (see Results). Others studying human chorionic gonado¬ trophin (Pollak, Halpine, Chait & Birken, 1990) have suggested that oxidation of methionine residues might give rise to hormone microheterogeneity. While this may be possible, its contribution in the case of ovine and bovine gonadotrophins is unlikely to be very sig¬ nificant in view of our above observations as well as the fact that solutions of the hormones (pH 7-5) kept at room temperature under sterile conditions for 3 weeks remained fully active.…”