M. T. W. Hearn

Isolation of inhibin from bovine follicular fluid

Bovine follicular fluid was used as a source for the isolation of gonadal inhibin, the activity of which was monitored by the dose dependent suppression of the FSH content of cultured pituitary cells. The procedures presented result in over 3000-fold purification of the starting material and the purified inhibin has an apparent molecular weight of 56000. The purified inhibin can be dissociated under reducing conditions into two subunits with molecular weights of 44000 and 14000 daltons.

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.

Isolation of a 31 kDa form of inhibin from bovine follicular fluid

The introduction of a pH 4.75 precipitation step to a previously described purification procedure from bovine follicular fluid (bFF) resulted in the isolation of a 31 kDa form of inhibin, in addition to 58 kDa inhibin. The procedure was monitored by an in vitro bioassay based on the suppression of the FSH cell content by pituitary cells in culture. The 31 kDa form was purified 5550-fold with approximately 5% recovery. On SDS-polyacrylamide gel electrophoresis a single band was detected with a molecular weight of 31 000 +/- 1500 (mean +/- SD) which upon reduction gave 2 subunits of 20 200 +/- 300 and 14 800 +/- 600. The biological activity expressed on mg protein basis was similar for both 31 kDa and 58 kDa inhibin although on a molar basis the 58 kDa inhibin was 2-3 times higher. A high degree of cross-reaction was observed between both forms in a radioimmunoassay of bovine inhibin using an antiserum raised against the larger form with either iodinated 31 kDa or 58 kDa inhibin as tracer. Based on the subunit composition of the 31 kDa and 58 kDa inhibin, their similar cross-reaction in a radioimmunoassay system and the apparent generation of the 31 kDa inhibin following a pH precipitation step, it is concluded that 31 kDa inhibin is a smaller form of the 58 kDa inhibin resulting from a shortening of the 43 kDa subunit to a 20 kDa subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography of amino acids, peptides and proteins: LXVII. Evaluation of bandwidth relationships of peptides related to human β-endorphin, separated by gradient-elution reversed-phase high-performance liquid chromatography

The bandwidths of several polypetides related to human β-endorphin have been investigated with different n-alkylsilica stationary phases and different elution gradients of 0.1% trifluoroacetic acid—water—acetonitrile mobile phases. In particular, we have examined the influence of changes in the gradient steepness parameter, b, on peakwidth with five different octadeccylphases, chemically bonded to porous spherical silica particles of nominally 4 μm and 6 μm average particle diameter, respectively. The effect on the zone dispersion of these polypetide solutes as the average more diameter of the silica matrix was increased from 7.3 nm to 30 nm with stationary phases of similar ligand densities packed into columns of identical configuration has been further documented. The experimental data on solute bandwidths and peak capacities are comparable with the corresponding bandwidth and peak capacity values calculated from analytical equations, derived from the general plate height theory and from gradient elution theory. These comparisons clearly demonstrate that anomalous bandbroadening phenomena may occur when polypeptides are eluted with steep gradients, i.e. with gradients of large b values. Moreover, as the relative chromatographic residence times of B-endorphin peptides capable of forming a C-terminal amphiphilic secondary structures is increased, i.e. as the dwell times and median capacity factors, k¯ for such peptides are increased, significant divergencies arise between the observed peakwidth behaviour and the behaviour predicted by analytical relationships which describe ether the dependency of peak bandwidth (as 4σv) on the gradient steepness parameter, b, or the dependency of peak capacity on gradient time, tG, median capacity factor, k¯, and the Knox parameter, C, respectively. The importance of these divergences from the predicted bandwidth and peak capacity behaviour for polypeptides separated on reversed phases, and for resolution optimisation in particular, is evaluated. These investigations thus enable further assessment of the quantitative relevance of current models that describe polypeptide zone migration under gradient elution reversed-phase chromatographic conditions in which solute-dependent slow equilibria, mediated by conformational or solvation effects, mayu still occur.