Abstract
A procedure for the isolation of bovine Factor V has been developed. The Factor V is isolated from bovine plasma by a series of steps including barium citrate adsorption, polyethylene glycol precipitation, QAE-cellulose adsorption, hydrophobic chromatography on octyl Sepharose, ammonium sulfate fractionation, preparative electrophoresis on acrylamide gels, and finally, phenyl Sepharose chromatography. During isolation, judicious use of inhibitors including benzamidine hydrochloride, soybean trypsin inhibitor, and diisopropylphosphorofluoridate has been applied to prevent activation of the Factor V TO Factor Va. The activity of the isolated protein increases by a factor of 80 when stimulated by catalytic amounts of thrombin. The specific activity of the material after thrombin activation is 1250 units/mg of protein when evaluated versus a bovine Factor V standard in human factor V-deficient plasma. The isolated protein is a single component when analyzed by a variety of electrophoretic techniques and has been characterized in terms of its gross physical and chemical properties. Bovine Factor V is a single chain glycoprotein which has a molecular weight of 330,000. The single chain nature of the molecule has been established by sedimentation equilibrium studies of the native molecule and on the molecule in 6 M guanidinium chloride with and without disulfide bond reduction. In addition to these mass measurements, the single chain nature of the molecule has been established by hydrodynamic estimation of the random coil volume by sedimentation velocity studies of the reduced carboxyamidomethylated protein in 6 M guanidinium chloride. Native Factor V has a sedimentation coefficient so20,w of 9.19 S, which indicates the molecule is highly asymmetric. The frictional ratio f/fmin for the molecule is estimated to be 2.01, and the axial ratio of the equivalent prolate ellipsoid is 25:1. Thus, present data suggest that Factor V is a rod-like molecule composed of a single chain.
Highlights
Factor V was eluted upon application of 500-ml linear gradient beginning with buffer plus 0.1 M NaCl and ending in buffer only
The isolation procedure described in this paper yields bovine Factor V which appears to be homogeneous when analyzed by a variety of techniques including gel electrophoresis, sedimentation equilibrium, and sedimentation velocity
The procedure provides overall yields of about 20%, or 4 to 5 mg of Factor V per liter of starting plasma, and has proven to be very reproducible, in that material of the same purity, specific activity, and activatibility by thrombin is obtained from preparation to preparation
Summary
The goal of the present work was to isolate and study the putative procofactor
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