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Journal of Lipid Research, Vol 28, 1508-1514, Copyright © 1987 by Lipid Research, Inc.

ARTICLES

Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity

PD Roach and SP Noel
Departement de Biochimie, Universite de Montreal, Canada.

Low density lipoproteins were biotinylated via free amino groups using carbodiimide-activated biotin or D-biotin-N-hydroxysuccinimide ester. The receptor binding activity of the biotinylated LDL was determined by their ability to displace 125I-labeled LDL from the rat hepatic LDL receptor in the liposome filtration assay. LDL biotinylated with either of the two reagents was able to compete effectively with 125I-labeled LDL providing less than twenty biotin moieties were incorporated per lipoprotein particle. When more than twenty biotins were linked there was a marked loss of activity. The following conditions were adopted as standard for the biotinylation of LDL via free amino groups: 0.3 mumol of D-biotin-N-hydroxysuccinimide ester was incubated with 2 mg of LDL for 1 hr at room temperature. These conditions reproducibly yielded 11.3 +/- 0.6 biotins per LDL particle. With the biotinylated LDL and a performed streptavidin-biotinylated peroxidase complex, the hepatic LDL receptor from rats treated with 17 alpha-ethinyl estradiol was visualized as a single band on electroblots. Finally, the biotinylated LDL was used in an enzyme-linked sorbent assay for the LDL receptor. When solubilized liver membrane proteins from rats treated with 17 alpha-ethinyl estradiol were fixed to the wells with 1.6% paraformaldehyde, a specific binding greater than 0.4 absorbance units was observed which was about ninefold higher than with solubilized proteins from normal rats. We therefore suggest that D-biotin-N- hydroxysuccinimide ester can be used to biotinylate LDL reliably without destroying the lipoprotein's ability to bind specifically to its high affinity receptor.
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				K. Berneis, M. La Belle, P. J. Blanche, and R. M. Krauss Analysis and quantitation of biotinylated apoB-containing lipoproteins with streptavidin-Cy3 J. Lipid Res., July 1, 2002; 43(7): 1155 - 1159. [Abstract] [Full Text] [PDF]