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JBC, Vol. 254, Issue 2, 508-517, Jan, 1979
M. E. Nesheim, K. H. Myrmel, L. Hibbard and K. G. Mann
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.
Isolation and characterization of single chain bovine factor V
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