Preparation and Certain Properties of Highly Purified Streptokinase

• Published in: The Journal of biological chemistry 1967-02, Vol.242 (3), p.533-542
• Main Authors: De Renzo, E C, Siiteri, P K, Hutchings, B L, Bell, P H
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Chromatography; Electrophoresis; Molecular Weight; Streptokinase - analysis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)96306-4

Procedures are described for the preparation of highly purified streptokinase by column chromatography on diethylaminoethyl cellulose and by column electrophoresis in a sucrose density gradient. Preparations chromatographed at least twice on diethylaminoethyl cellulose are shown to be essentially monodisperse on the basis of ultracentrifugal and gel electrophoretic analyses and constancy of specific activity. In good agreement with a previous finding from this laboratory, the molecular weight determined by equilibrium sedimentation was found to be 47,600. Treatment of streptokinase at pH 7.5 in 0.1 m phosphate buffer with 5 m guanidine-hydrochloride or 8 m urea produced a lowering of the sedimentation coefficient without significantly changing the molecular weight. Cystine and cysteine were absent on amino acid analysis, and the molecule is therefore assumed to consist of a single polypeptide chain with no subunits. The isoelectric point was found to be about pH 4.7. The amino acid composition is consistent with the formula Asp 68 -Thr 30 -Ser 24 -Glu 46 -Pro 20 -Gly 21 -Ala 23 -Val 23 -Met 3 -Ile 22 Leu 40 -Tyr 20 -Phe 15 -Lys 33 -His 9 -Arg 21 -Try 1 for molecular weight 47,754. Approximately 60 aspartic and glutamic residues are amidated per molecule of protein. The most highly purified preparations are devoid of carbohydrate and phosphorus and are inactive with basic amino acid esters, naphthyl esters, and acetyltyrosine ethyl ester as substrates.