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When plasma containing a hepatitis-associated antigen (Au/SH) is fractionated, the antigen is localized in fractions III and IV with none in fraction II and only small amounts in fractions I and V. The amount of antigen found in each of these fractions is probably not predictive of clinical infectivity of Cohn ethanol fractions from normnal pooled plasna.

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Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.-1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VI.The most common inherited bleeding disorders are associated with deficiencies or defects in factor VIII or von Willebrand protein. Much of the physiological role and structural features of the von Willebrand protein have been characterized (1); much less is known about factor VIII. Two major factors have hindered the study offactor VIII-the low concentration of factor VIII in plasma, and the difficulty of obtaining a purified, homogeneous preparation. Recent reports have described a high degree ofpurification and some characterization of bovine factor VIII (2), porcine factor VIII (3), and human factor VIII (4). However, the final preparations contained several polypeptide chains when analyzed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis under reducing conditions. We report here a procedure for purification of human factor VIII from plasma which utilizes calcium dissociation and differential size and charge chromatography. The (8). The source ofvon Willebrand protein used for the enzyme-linked immunosorbant assay was the void volume fraction eluted from Sepharose CL-4B obtained during the purification of factor VIII. Hemophiliac plasma was obtained from a patient with severe factor VIII deficiency as shown by a glass clotting time >60 min (9). The factor VIII inhibitor plasma used in this study had a titer of 6,400 Bethesda units. BioGel A-15m, Sepharose CL-4B, and QAE cellulose (fine mesh) were purchased from Bio-Rad Laboratories, Pharmacia, and Sigma, respectively. The protein silver staining kit was purchased from Bio-Rad.Assays. Protein concentration was determined by absorbance at 280 nm corrected for light scattering; an E" corrected of 10.0 for factor VIII was assumed. Factor VIII procoagulant activity was quantitated by a one-stage clotting assay (10). In the presence of0.25 M CaCl2, factor VIII procoagulant activity was measured by using a modified one-stage assay (11). Factor VIII antigen was measured according to the procedure of Reisner et al. (12); 1 unit of factor VIII antigen r...

Immune serum globulin (ISG) prepared by Cohn cold alcohol fractionation of pooled human plasma was reduced with dithiothreitol (DTT) and alkylated with iodoace- tamide and other alkylating agents. Our results show that there are a few labile interheavy chain disulfide bonds in ISG which react rapidly under mild, nondissociating conditions. The extent of disulfide cleavage is controlled primarily by the ratio of DTT to ISG until about 4-5 disulfide bonds have been reduced. We report detailed studies on the variables of ISG concentration, DTT to ISG ratio, pH, and time, leading to a chemically modified ISG that has a controlled and limited number of reduced and alkylated disulfide bonds.