Abstract
Urinary gonadotrophin peptide (UGP) was originally identified by immunoassay in the urine of patients with various types of cancer and by immunohistochemistry in human cancers of various histological types. Extracts of normal adult male urine also contained UGP by immunoassay. Purified UGP from different starting material was subjected to high pressure liquid chromatography (HPLC) prior to defining amino acid sequences. Chromatographed UGP after HPLC showed three distinct fractions. The N-terminal sequence of peptide 2 was completely homologous with the beta-core fragment of human chorionic gonadotrophin (hCG) and this was found associated with two smaller peptides. The N-terminal sequence of peptide 1 has not been described previously whilst the N-terminus of peptide 3 that was sequenced showed complete homology with the N-terminal sequence of eosinophil derived neurotoxin and non-secretory ribonuclease. The monoclonal antibodies 2C2 and 6D3 only bind beta core-fragment (peptide 2) whilst the polyclonal (rabbit) antibody AK12 could bind all three peptides. The radioimmunoassay system using AK12 could be inhibited by all three peptides and the immunoradiometric assay although based on a capture antibody (2C2) that only bound peptide 2, had the potential to measure all three peptides (when bound together as UGP) at the second step when 125I-AK12 was introduced as the detector. A specific radioimmunoassay for peptide 3 was generated using 125I-peptide 3 and the AK12 antibody. Beta core-fragment on iso-electric focusing was found to have a pI > 9.5, peptide 3 showed two bands at pI = 3.5 and 3.8 whilst insufficient purified peptide 1 was available to determine its iso-electric point. Bioassay studies on UGP showed that any biological activity could be attributed to trace contamination with hCG.
Highlights
In this paper we report further characterisation of Urinary gonadotrophin peptide (UGP) using high pressure liquid chromatography, amino acid sequencing and iso electric focusing
Pregnancy UGP purified by gel-filtration, antibody affinity chromatography rechromatographed on a Superose 12 column, as well as purified UGP from the three other sources was subjected to high pressure liquid chromatography (HPLC) separation (Figure 1)
Amino acid sequence data was obtained for fractions 1, 2 and 3
Summary
Purification of UGP A commercial preparation of hCG (Pregnyl) derived from pooled pregnancy urines was subjected to gel filtration on Sephadex G 100 and immunoadsorption with a mouseCorrespondence: F. Purification of UGP A commercial preparation of hCG (Pregnyl) derived from pooled pregnancy urines was subjected to gel filtration on Sephadex G 100 and immunoadsorption with a mouse. Aliquots of the immunoadsorbed material were further purified on FPLC using a Superose 12 column (Pharmacia FPLC systems) or re-subjected to gel-filtration using Sephadex G-100. UGP (determined by immunoassay) from the first gel-filtration step, was loaded on to a Concanavalin A-Sepharose column (Pharmacia Fine Chemicals) that was equilibrated, eluted with Con A buffer and eluted with Con A buffer containing 0.5 M a-methyl-D-mannoside. The unbound fraction from DEAE chromatography was immunoadsorbed as above and desalted using Sephadex G 100 (2.5 x 80 cm column). The unbound material from Con A chromatography was immunoadsorbed and desalted on Sephadex G 100
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