Year: 1979
Type: article
Abstract: A procedure is described for the purification of human plasma prekallikrein, the precursor protein of the serine protease kallikrein. Human plasma was mixed for 30 min with an equal volume of preswollen quaternaryaminoethyl-Sephadex equilibrated in sodium phosphate (10 mM) buffer pH 8.0 containing hexadimethrine bromide, and then filtered. The filtrate was cycled two more times and the final filtrate was chromatographed on a sulphopropyl-Sephadex column at pH 8.0. The concentrated effluent was chromatographed on a sulphopropyl-Sephadex column at pH 5.2. The final step of purification was affinity chromatography on agarose coupled to antiserum to human IgG to remove contaminating IgG. A single peak of protein emerged coincident with prekallikrein antigen representing an overall yield of 39% of the starting material. After activation of the final product with purified Hageman factor fragments, kinin-releasing, arginine esterase and amidolytic activity developed. The activated enzyme hydrolyzed the amide bond in the synthetic substrate l-prolyl-l-phenylalanyl-l-arginine-p-nitroanilide with a Km of 0.225 mM. This 1800-fold purified prekallikrein appeared as a major and minor band on alkaline disc/polyacrylamide gel electrophoresis and each component was associated with prekallikrein coagulant activity. On dodecyl sulfate gel electrophoresis, a single band was observed with a Mr of 88000. When prekallikrein was activated by Hageman factor fragments to kallikrein, and this activated enzyme was subjected to reduction, two subunits with Mr of 55000 and 33000 were apparent.
Source: European Journal of Biochemistry
Institutions: Temple University, Columbia University
Language: English
Subfield: Genetics
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2089682872