Fixation of bovine casein micelles for chromatography on controlled pore glass

Summary

Glutaraldehyde was used to fix the size distribution of casein micelles in skim milk before their fractionation by permeation chromatography on controlled pore glass. The effect of fixation was assessed by comparing the size and absorption profile for column fractions obtained from samples which were fixed before fractionation with those of unfixed samples and of samples that were fixed only after completion of chromatography. Micelle size was determined by photon correlation spectroscopy. Column profiles were obtained from absorption measurements at 340 nm and after pronase digestion at 280 nm to determine relative protein concentration. For comparative purposes the elution profile was divided into 4 peak areas, of which I-III contained most of the casein micelles, and IV consisted of the smallest micelles, soluble casein and whey protein. Average micelle size was unaltered by fixation but was larger in fractions from prefixed skim milk than those from unfixed samples. Fixation increased the absorbance readings but the elution profile at 340 nm for peak areas I-III was essentially the same for unfixed fractions and those obtained when fixation was applied after chromatography. However, areas I-III accounted for a much larger proportion of the total profile area when the latter procedure was followed. Total profile area increased with fixation time and temperature but this did not affect the elution profile. The results indicate that fixation with glutaraldehyde does not induce artefactual changes in casein micelle size.