The activity of this material was, cmg.The pH of the milk was adjusted to with glacial acetic acid and the precipitated casein removed by centrifuging.The casein was washed twice with msodium acetate buffer at pH, and the washings were added to the whey.The combined washings and whey were saturated to with SO.The precipitated proteins were removed by centrifuging and washed with a saturated J solution.The supernatant fluid was treated with saturated ammonium sulphate solution and the following fractions were collected.The powder was dissolved in ml.The contents of the tubes with the bound cyanocobalamin were combined, and concentrated by ultrafiltration to a volume of ml.This concentrated solution at pH was fractionated with iopropanol at. Therefore the assessment of purity was based on the fact that the substance was homogenous when examined by electrophoresis on paper at both pH, and that the ratio of the concentration of cyanocobalamin to protein was unaltered by further precipitation with iopropanol.In a previous paper we have shown that an unheated protein concentrate from sows milk bound more cyanocobalamin than the heated concentrate.It was necessary, therefore, to devise some method for measuring the amount of cyanocobalamin bound by unheated whole milk.Natural inhibitors present in raw milk complicate the interpretation of the microbiological responses when unheated milk was added aseptically to the assay tubes. In the ultrafiltration method finally adopted, the actual binding process was separated from the microbiological assay.The technique consisted of mixing the milk or binding protein with a slight excess of cyanocobalamin, and ultrafiltering the mixture.By removing only a small proportion of the fluid by ultrafiltration, the equilibrium of the system was not appreciably disturbed and the concentration of cyanocobalamin measured in the ultrafiltrate represented the concentration of the free vitamin in the whole system.When the amount of cyanocobalamin bound by a sows whey concentrate was measured by this ultrafiltration method, the result was identical with that obtained M.It has also shown that the milk and colostrum of other animal species have the <a href="http://www.targetmol.com/compound/Reboxetine">buy
Reboxetine</a> capacity of binding additional cyanocobalamin, a property not detected when the milks were heated with the basal assay medium.Microbiological assays and autoradiographs of milk extracts, after separation by electrophoresis on paper showed that the bound form of the vitamin in the milks, and the complexes formed when the vitamin was added to the milks, were very similar if not identical.Moreover, the bound form of cyanocobalamin from milk had the same mobility as the cyanocobalaminprotein complex isolated from desiccated pig stomach.The different heat stability of the complex in these milks makes it impossible to state that the milks all contain the same bound form of cyanocobalamin.Earlier experiments showed that the substance responsible for inactivating cyanocobalamin was not one of the major components of sows whey. Attempts to isolate the binding factor from sows whey were unsuccessful because of considerable losses during fractionation and a more satisfactory procedure was to saturate the binding factor with radioactive cyanocobalamin and isolate the more stable complex by following the radioactivity.We have shown that cyanocobalamin occurs in a bound form in milk from different animal species.