Isolierung und Charakterisierung von sialinsäurehaltigem und sialinsäurefreiem Kallikrein aus Schweinepankreas

Abstract

Adsorption chromatography on hydroxylapatite-cellulose columns gives an excellent purification of kallikrein from pig pancreas. Partially purified kallikrein preparations were chromato-graphed 3 times on hydroxylapatide; the yield of kallikrein was 50-60% with a specific activity of 0.70-0.75 kallikrein units or 133-143 enzyme units/mg of biuret protein. The amino acid composition and sugar content of kallikrein were determined. Besides glucosamine (3.0-3.3%) and galactose (0.7%), the presence of mannose (2%), fucose (1%), glucose (0.3%) and sialic acid (0.8%) was demonstrated for the first time. Electrophoresis at pH 4-5 shows that the isolated kallikrein consists of a mixture of 2 kallikreins that contain no sialic acid and several sialic acid-containing fractions. The latter constituted 30-50% of the mixture. Treatment with neuraminidase almost completely removes the sialic acids. Electrophoresis then only shows 2 kallikreins, which contain sialic acid. The separate fractions have essentially the same specific activity and the same action on the blood pressure of the dog. The composition analysis (283 amino acid residues and 19-21 sugar residues) gives a molecular weight of 33,400-33,800 for kallikrein containing no sialic acid. The molar extinction coefficient of an aqueous solution of kallikrein is 5.1 x 104 at the maximum absorption wavelength (282 m[mu]). UV-spectra of kallikrein, made under different conditions, are discussed. The stability of kallikrein in weakly acidic and weakly alkaline solutions was studied.