Purification and characterization of bovine parotid hypocalcemic factor obtained via extraction with glacial acetic acid.

Abstract

The acetone-dried powder (PA), obtained from supernatant left after separation of the pH 5.4-precipitating fraction (crude Parotin) from the aqueous extract of bovine parotid gland, was extracted with glacial acetic acid, and the extract had a hypocalcemic activity in rabbits, but its activity fluctuated. On the other hand, the glacial acetic acid extract of the acetone-dried powder (PAI) obtained by incubation of a suspension of the sample PA in saline, had nearly constant activity. From the purification of PAI by DEAE-cellulose chromatography and gel chromatography on Sephadex G-100, a hypocalcemic protein (P-MSY) was obtained in a yield of 37 mg from 10 kg of the fresh gland. The sample P-MSY was homogeneous in disc electrophoresis and electrofocusing, and it was effective in a dose of 0.01 mg/kg at 1% level of significance against control rabbits given saline, and it has almost the same activity as purified Parotin, which was effective in a dose of 0.01 mg/kg as reported previously. P-MSY had a molecular weight of 66000 by SDS-polyacrylamide gel electrophoresis, isoelectric point of pH 5.60, and sugar content of 2.53%. These properties and the results of amino acid analysis suggest that P-MSY is a hypocalcemic protein differing from purified Parotin. Further, P-MSY was also proved to be different from β-Parotin or calcitonin in its character.