Urokinase: Isolation, Purification, Characterization, New Spectrophotometric Bioassay Method and In-vitro Blood Clot Dissolving Activity from Cow Urine

摘要

Urokinase, a potent plasminogen activator is an enzyme present in cowurine attributing to the thromboembolic properties of cow urine but its presence in cow urine has never been convincingly documented. In our work, we did isolation, purification, partial characterization and quantitation of urokinase in cowurine using newly developed spectrophotometric bioassay method and studied its invitro blood clot dissolving activity. The cow urine was collected from cows of Indian origin ( Bos indicus ). Isolation of Urokinase was done by ammonium sulfate precipitation and dialysis. Purification was done by using Sephadex G-200 Gel Filtration Chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Gelatin Zymography, HPLC and TLC of tryptic digest of the purified urokinase was carried out for partial characterization studies which conformed the molecular weight of 59 kD of the purified Urokinase. Quantification with the help of new spectrophotometric bioassay method showed 956 IU/ml and with the help of HPLC it was 910 IU/ml of urokinase in cow urine. The invitro blood clot dissolving activity on a cotton cloth confirmed the activity and efficacy of the isolated and purified urokinase.