Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.
Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.
Hermann
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Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
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Harpel
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D000578:Amidines; D001053:Apolipoproteins / Q000737:chemistry / Q000378:metabolism*; D053241:Apoprotein(a); D001665:Binding Sites; D002839:Chromans; D005453:Fluorescence; D018082:Kringles; D017270:Lipoprotein(a); D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D000578:Amidines; D001053:Apolipoproteins; D002839:Chromans; D017270:Lipoprotein(a); C046728:2,2'-azobis(2-amidinopropane); D014364:Tryptophan; D053241:Apoprotein(a); D008239:Lysine; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid