Attenuation of immunologic reactivity of lipoprotein(a) by thiols and cysteine-containing compounds. Structural implications.
Samples of human plasma having lipoprotein(a) (Lp[a]) protein levels between 5 and 15 mg/dl and a single apolipoprotein(a) (apo[a]) isoform were incubated in vitro at pH 7.7 with various concentrations (1-20 mM) of N-acetylcysteine, homocysteine, 2-mercaptoethanol (2ME), and dithiothreitol (DTT) for 1 hour at 37 degrees C under a nitrogen atmosphere. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analyses using a polyclonal antibody specific for apo(a) showed a progressive decrease in apo(a) immunoreactivity as a function of reductant concentration. This decrease of apo(a) immunoreactivity was corroborated by enzyme-linked immunosorbent assay (ELISA) using anti-apo(a) as the capture antibody and either anti-apo B or anti-apo(a) as the developing antibody. In turn, there was no significant decrease in the immunoreactivity of apo B-100, as assessed by ELISA using anti-apo B as both the capture and the detecting antibody. In the case of high concentrations of DTT the plasma samples had to be diluted to prevent gel formation on addition of the reductant. A progressive drop in immunoreactivity as a function of reagent concentration was also observed in pure preparations of Lp(a) incubated with the reducing agents at pH 7.7. At equivalent stoichiometries the changes were more marked than those observed with whole plasma, suggesting a quenching effect by the plasma proteins on the activity of the reductants. The changes in immunoreactivity were attended by dissociation of apo(a) from Lp(a) as assessed by Western blotting. This dissociation, which we interpret as the result of cleavage of the interchain disulfide bond(s), was complete at 5 mM DTT and 100 mM 2ME.(ABSTRACT TRUNCATED AT 250 WORDS)
Scanu
A M
AM
Department of Medicine, University of Chicago, IL 60637.
Pfaffinger
D
D
Fless
G M
GM
Makino
K
K
Eisenbart
J
J
Hinman
J
J
D000111:Acetylcysteine / Q000494:pharmacology; D001054:Apolipoproteins A / Q000276:immunology; D001055:Apolipoproteins B / Q000276:immunology; D015153:Blotting, Western; D003545:Cysteine / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D017270:Lipoprotein(a); D008074:Lipoproteins / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D015394:Molecular Structure; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D001054:Apolipoproteins A; D001055:Apolipoproteins B; D017270:Lipoprotein(a); D008074:Lipoproteins; D013438:Sulfhydryl Compounds; D006710:Homocysteine; D008623:Mercaptoethanol; D003545:Cysteine; D004229:Dithiothreitol; D000111:Acetylcysteine