Determination Of Molecular Size Of VIII:C In Whole Plasma By Electron Irradiation
Loss of activity during electron irradiation provides a means of determining the molecular size of specific molecules in complex biological mixtures. This technique has established a molecular weight of 200,000 for Factor VIII:C in concentrates prepared from citrated plasma in which calcium is sequestered by chelation (Aronson, et al. Thromb. Diath. Haemorrh. 8:270, 1962): this value is similar to that obtained by more conventional techniques. However recent data suggest that in heparinized plasma, where physiological levels of calcium are maintained, about half of the VIII:C activity has a molecular weight of 50,000 as determined by gel filtration and ultracentrifugation (Rock, et al. Thromb. Res. 13:85, 1978). When heparinized plasma was subjected to electron irradiation in the frozen state there was a perceptible loss of VIII:C activity at 1 megarad and 80% loss with 60 megarads of irradiation. Analysis of the course of inactivation showed a biphasic curve with 73% of the VIII:C activity having a target size of 40,000 daltons while 28% had a molecular weight in excess of one million. Similar results were obtained when blood was collected in citrate and rapidly processed (∼5 min) to platelet-poor plasma. Following electron irradiation, 62% of VIII:C activity showed a target size of 35,000 daltons while the remaining 38% gave a target size of 275,000. These results provide further evidence that circulating VIII:C activity in normal plasma has a molecular weight of about 40,000 and suggest that reports of higher molecular weights are an artifact of the chelation of calcium as evidenced by the biphasic decay of VIII:C activity in citrated plasma.