Mean Molecular Weights of Synthetic Mixtures of Bovine Plasma Albumin and Globulin

Abstract

Albumin and globulin, though generally considered to be separate entities, show sedimentation anomalies when they are studied in mixtures with the ultracentrifuge (Pederson 1 ). The data of McFarlane 2 suggest that globulin may dissociate in such mixtures. We have studied purified bovine plasma albumin and globulin, alone and in synthetic mixtures, by osmometric methods.

Sterile bovine plasma was carefully fractionated with ammonium sulfate at 0°C by a technic similar to that used by McMeekin. 3 The albumin and globulin fractions were reprecipitated, freed of ammonia by dialysis and then dialyzed to equilibrium with phosphate buffer of pH 7.4, μ = 0.16. Osmotic pressures were measured at 0°C by the method to be described by Keys. Protein concentrations were determined with the Pulfrich refractometer in every sample and checked by the micro-Kjeldahl method of Keys 4 in about every third sample. Mean molecular weights were determined by the method of Adair and Robinson 5 in which the pressure-concentration ratio is extrapolated at zero concentration. At least 4 acceptable P/C values at below 3% protein concentration were obtained for each sample.

The molecular weights of the albumin and globulin used here were 69,900 and 170,000, respectively, and these values were used to compute the theoretical mean mol. wts. of the mixtures from Dalton's law of partial pressures. The observed mol. wts. of the mixtures deviated widely and systematically from the simple predictions. As shown in Fig. 1, the mean mol. wts. of the mixtures are lower, and the osmotic pressures are higher than predicted. The deviation approaches a linear function of the mol. wt. in the range of A/G from 0.1 to 2.1. Similar results were obtained with other preparations.

Sedimentation diagrams of such mixtures clearly show 2 fractions corresponding in molecular size with albumin and globulin but McFarlane's (op. cit.) data on horse plasma proteins indicate that the proportion of the smaller molecules is higher than the composition of his mixtures would warrant.