Half of the enzyme was added at the beginning of the experiment and the remainder halfway through the incubation period.The remainder of each digest was ultrafiltered and the vitamin B activity of the ultrafiltrates measured.Both the digest and ultrafiltrate were assayed without further treatment other than dilution with water.Digestion of the binding factor with papain, trypsin or pepsin reduced its ability to combine with added cyanocobalamin and from to of the added vitamin appeared in the ultrafiltrate. After digestion with chymotrypsin and carboxypeptidase the binding activity of the sowswhey factor was also reduced, but to a lesser extent than with the other enzymes.However, when the binding factor was saturated with cyanocobalamin before treatment with the enzymes it was more resistant to their attack.Therefore the enzymes were not able to release free cyanocobalamin from the binding protein, although they were able to increase the microbiological availability of the vitamin in the complex.Rough quantitative tests with dilutions of antigen were performed, by using various binding substances which had been saturated with radioactive cyanocobalamin and freed from excess by prolonged dialysis.When quantities of the different materials with equal binding capacities were tested against the antiserum, pigpylorus and sowsmilk antigens gave closely similar reactions.Other complexes showed crossreactions, but only at much higher concentrations.The precipitate was removed by centrifuging and washed with saturated ammonium sulphate solution.Since no radioactivity was detected in the precipitate, it was rejected.A similar precipitate obtained at saturation had only slight radioactivity and was also discarded.Proteins remaining in solution were precipitated by adding solid ammonium sulphate to saturation, again at. The solution was refractionated with ammonium sulphate and saturation yielded the crude cyanocobalamin complex.After dialysis against distilledwater the protein solution, and cooled to and propanol at was added dropwise with stirring.The temperature was gradually lowered to during the fractionation.Thevarious fractions obtained were tested for the presence of cyanocobalamin by dissolving in water and measuring the radioactivity. The resulting pink protein was dissolved in water and dialysed against changes of distilled water.The solution was cooled to and fractionated with acetic acid in propanol, when the pink material precipitated at a <a href="http://www.targetmol.com/compound/Ribavirin"></a>
concentration of between and of the acid alcohol.Alternate precipitations at pH and with acid propanol were continued until the ratio of the extinction coefficient at mpt reached a steady value.After dialysis against distilled water the complex was obtained as a pink powder by freezedrying the solution. The material migrated as a single pink band, with a mobility close to that of xlglobulin, on paper electrophoresis at pH. It differed from ocglobulin in its solubility properties and did not stain on paper with bromophenol blue.As far as could be ascertained the chemical properties resembled those of the two complexes examined previously, one from sows milk. Thus the complex is a glyco or mucoprotein.Each group concluded that the vitamin B was associated mainly with the al and ocglobulin.This distribution between different fractions can be explained if the vitamin was combined with a minor component whose properties overlap those of the main serum proteins, and since the papers were cut into these main fractions the vitamin activity would be spread over a number of them.