MOLECULAR-PROPERTIES OF LIPOPROTEIN-LIPASE - EFFECTS OF LIMITED TRYPSIN DIGESTION ON MOLECULAR-WEIGHT AND SECONDARY STRUCTURE

Abstract

The monomer molecular size of bovine lipoprotein lipase was evaluated by sedimentation equilibrium measurements and by gel permeation chromatography in 6 M guanidinium chloride. To establish MW unequivocally the partial specific volume (.hivin.v) was determined. This was done by analyzing equilibrium concentration profiles from analytical ultracentrifugation in 6 M guanidinium chloride using buffers made up in H2O and 2H2O. The combined results gave a .hivin.v of 0.71 .+-. 0.007 ml/g and a MW of 41,700 .+-. 1000 for monomeric bovine lipoprotein lipase. This value did not change upon mild tryptic digestion; the elution volume upon gel permeation chromatography in 6 M guanidinium chloride was also unaffected by treatment with trypsin. Sedimentation equilibrium measurements of the trypsin-treated material in the presence of reducing agents gave limiting MW of 19,000 and 23,000, demonstrating that mild trypsin digestion cleaved lipoprotein lipase into 2 polypeptide chains of similar size held together by S-S bonds. Mild trypsin digestion also resulted in a loss of secondary structure as determined by circular dichroic measurements. Discussion centers around the correlation between these effects of trypsin on the molecular properties of lipoprotein lipase and previously reported effects on the kinetic properties of the enzyme.