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In a previous communication from this laboratory (Stockell Hartree, Thomas, Braikevitch, Bell, Christie, Spaull, Taylor & Pierce, 1971) it was reported that the sialic acid contents of the subunits of human luteinizing hormone (LH) were very much less than in the intact hormone. This finding was unexpected since the subunits were still capable of recombination with restoration of full biological activity. The demonstration that marked loss of biological activity occurred in human LH after removal of sialic acid by treatment of the intact hormone with neuraminidase (Braunstein, Reichert, Van Hall, Vaitukaitis & Ross, 1971) has prompted us to reinvestigate the sialic acid contents of the subunits. In addition, the tryptophan content of the subunits has been measured by the spectrophotometric method of Bencze & Schmid (1957).
Estimations were performed on subunits prepared by two different methods. In each case intact LH of potency 3–5 mg NIH-LH-S1/mg (Stockell Hartree, 1966) was
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Procedures for purification of human luteinizing hormone (LH) and human folliclestimulating hormone (FSH) (Stockell Hartree, 1966; Roos, 1968) have been applied to acetone-preserved baboon pituitaries. After death of the baboons in Kenya pituitaries were removed and placed in excess cold acetone. The glands were transported to Ohio where an acetone-dried powder was prepared, the yield from 185 pituitaries was 10·1 g. This material was extracted with 6% ammonium acetate at pH 5·1 in 40% ethanol and protein was precipitated from the soluble extract by addition of ethanol to 80% (GTN ppt). This fraction was then chromatographed on a column of CM-cellulose as described previously (Stockell Hartree, 1966). The nonadsorbed fraction (CM-1) was pooled, concentrated by rotary evaporation and freeze-dried. The adsorbed fraction (CM-2) was eluted with 1 m-ammonium acetate, and was precipitated with 80% ethanol. Fraction CM-2 (44 mg) was chromatographed on a column of DEAE-cellulose (Whatman DE-23, 1
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SUMMARY
Two methods for the purification of corticotrophin from human pituitary glands have been assessed by two radioimmunoassays directed towards the C-terminal and N-terminal parts of the molecule, and by bioassay. One procedure, applied to small amounts of tissue, employs acid acetone extraction, oxycellulose adsorption and chromatography on CM-cellulose (CM-23). It is suitable for the small-scale purification of corticotrophin from pituitary glands but the losses associated with extracting low concentrations of hormone suggest that it would be inadequate for application to extrapituitary tissues and tumours. The starting material for the second procedure was a side fraction derived from the large scale extraction of protein hormones from whole pituitary glands. The purification employs oxycellulose adsorption, gel filtration on Sephadex G-50 and chromatography on CM-cellulose (CM-32). It yields sufficient highly potent corticotrophin for use as radioimmunoassay standard while conserving other scarce human pituitary hormones.
