Sulfhydryl compounds influence immunoreactivity, structure and functional aspects of lipoprotein(a)

Abstract

Human plasma Lp(a) is susceptible to various sulfhydryl compounds. In this study we present evidence indicating that after treatment of Lp(a) with sulfhydryl compounds, immunoreactivity is changed, structural changes occur and functional characteristics regarding the numerous kringle structures in apo(a) disappear. Purified Lp(a) was subjected to variable concentrations (0.01-10 mM) of various sulfhydryl compounds: DTT, 2-mercapto-ethanol (BME), N-acetylcysteine (NAC) and homocysteine (HCys). Free SH groups were blocked by iodoacetamide. Reduced and alkylated Lp(a) was tested in two ELISAs, one detecting apo(a) alone and one detecting apo(a)-apoB complexes. In both ELISAs polyclonal antibodies were used. For comparison a commercial apo(a) IRMA utilizing two monoclonal antibodies was used. The results indicate that a similar decrease in response of both ELISAs is observed, whereas the IRMA response is less affected. Western blotting of "DTT treated" Lp(a) after SDS-PAGE under nonreducing conditions showed that separate apo(a) and apoB-100 bands became detectable at 1 mM DTT. Native PAGE (2.5-16%) indicated structural changes of Lp(a) beginning to occur at 0.03 mM DTT. Epsilon-aminocaproic acid-inhibitable binding of "DTT-treated" Lp(a) to Desafib-X decreased with increasing DTT concentrations in concert with a loss of the capacity of Lp(a) to inhibit plasminogen activation upon treatment with DTT. The observed immunological and functional changes of Lp(a) indicate that apo(a) kringle function is severely affected by sulfhydryl compounds.