Application of high-performance liquid chromatography to determination of modifier activity in alpha-lactalbumin and...

High-performance liquid chromatography has been applied to determination of modifier activity in alpha-lactalbumin (alpha-LA). An amino-bonded column separates uridine diphosphate (UDP) (product), UDPgalactose (substrate), and uridine monophosphate (UMP). From an aliquot of the same sample, a column for carbohydrate analysis separates lactose (the other product) and galactose-1,2-cyclic phosphate (Gal-c-P). Nucleotide peaks are detected by measurement of A262 and those of carbohydrate by 3H counting, the isotope originating from UDP-galactose-3H. A pH of 6.3 was taken as optimal for production of UDP since, at this level, the unwanted side reaction is minimized, by which UMP and Gal-c-P are formed. Thus, the conservation of substrate so effected may have contributed to an enhanced production of UDP. The reaction by which UDP and lactose are produced was linear for 120 min, as followed by UDP formation, but it continued to at least 300 min. Production of lactose was equivalent to that of UDP, when alpha-LA was the modifying protein. From a survey of seven other proteins, only lysozyme and ovalbumin showed ability to produce UDP. However, failure of the last two proteins to produce lactose indicates absence of modifier activity and demonstrates the need for monitoring both products.