Physico-chemical characterization of growth hormone from water buffaloes (Bubalus bubalis)

Abstract

A purified preparation of growth hormone from pituitaries of water buffaloes (Bubalus bubalis) has been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00 ' 8.44Da and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by electrophoresis and molecular sieve chromatography using125I-labelled buffalo GH. Similar size heterogeneity was found in standard preparations of ovine and bovine growth hormones. Isoelectric focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge isoforms having pl of 7.2, 7.7 and minor forms in the pl range of 5.7 to 7.0 were found. Lectin chromatography on Concanavalin A matrix showed that less than 1% of buffalo GH was glycosylated. Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine as the NH²-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan residue indicated that a single tryptophan residue was present. Ellman's method showed presence of two disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited X emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of buGH was constituted by α-helicity. The TM of buGH as determined by differential scanning calorimetric (DSC) studies was found to he 63°C.