Identification of a contaminant protein in the purification of human factor V. Comparison of two purification methods

Coagulation factor V has been isolated from human plasma by the methods of Bolhuis and of Kane to investigate reported differences in purity and specific activity. The specific activities of the preparations are 10 and 40 units/mg, respectively. The Kane preparation shows a single band (Mr 330 000) in polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, but the Bolhuis preparation shows an additional band at Mr 190 000 upon reduction with dithiothreitol. This suggests the presence of a contaminant which could account for the lower specific activity. Modification of the Bolhuis technique to include ion-exchange chromatography on DEAE-Sepharose resulted in separation of the two isolated proteins. The proteins were identified as factor V, with characteristics indistinguishable from Kane factor V, and a dimeric subunit of alpha 2-macroglobulin (Mr 350 000). Contamination by alpha 2-macroglobulin may explain the I2-component described in some preparations of bovine factor V, which is the basis of speculation that bovine factor V has a second polypeptide chain modulating stability and activity.