Open tubular capillary electrochromatography: a new technique for in situ enzymatic modification of low density lipoprotein particles and their protein-free derivatives

Abstract

Electrochromatography with open tubular capillaries coated with human low density lipoprotein (LDL) particles and their protein-free derivatives was studied as a method for their in situ enzymatic modification. LDL particles as monolayers or their protein-free derivatives (lipid microemulsions) were coated on 50 µm i.d. capillaries, which resemble tiny human blood vessels in size, the arterioles. The immobilized LDL particles were exposed to sphingomyelinase, phospholipase A₂ or α-chymotrypsin at 25 and 37 °C. The mobility of the electro-osmotic flow was employed as a surface charge indicator, and the retention factors of steroids were used as hydrophobicity indicators. Moreover, the capillaries were, for the first time, coated with lipid microemulsions containing either LDL-derived or commercial lipids, and the immobilized microemulsions were treated with sphingomyelinase in capillary. The results demonstrate that open tubular capillaries provide a good microreactor for the in situ modification of LDL particles and lipid microemulsions. The technique only requires extremely low quantities of LDL particles, lipid microemulsions, and enzymes. It allows quick and easy alteration of the reaction conditions, and the enzymes can be collected and reused. Asymmetrical flow field flow fractionation provides useful information on the size of the enzymatically modified LDL particles.