Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity.
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.
Roach
P D
PD
Département de Biochimie, Université de Montréal, Canada.
Noël
S P
SP
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000032:analysis; D008297:Male; D008722:Methods; D051381:Rats; D011919:Rats, Inbred Strains; D011973:Receptors, LDL / Q000378:metabolism*
D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D001710:Biotin