Reagent or myeloperoxidase-generated hypochlorite affects discrete regionsin lipid-free and lipid-associated human apolipoprotein A-1

We have previously shown that the modification of high-density lipoprotein subclass 3 (HDL3) by HOCl transformed an antiatherogenic lipoprotein into a high-uptake form for macrophages and caused a significant impairment of ch olesterol efflux capacity [Panzenboeck, Raitmayer, Reicher, Lindner, Glatte r, Malle and Sattler (1997) J, Biol. Chem. 272, 29711-29720]. To elucidate the consequences of treatment with OCl- on distinct regions in apolipoprote in A-I (apo A-I), lipid-free and lipid-associated apo A-I were modified wit h increasing molar ratios of NaOCl or HOCl generated by the myeloperoxidase /H2O2/Cl- system. CD analysis revealed a pronounced decrease in alpha-helic ity for lipid-free apo A-I modified by NaOCl, whereas lipid-associated apo A-I was less affected. The modification of apo A-I by NaOCl (molar oxidant- to-lipoprotein ratio 6: 1) resulted in the formation of two distinct oxidiz ed forms of apo A-I with molecular masses 32 or 48 atomic mass units (a.m.u .) higher than that of native apo A-I, indicating the addition of two or th ree oxygen atoms to the native protein. HPLC analysis of tryptic digests ob tained from lipid-free and lipid-associated apo A-I modified with increasin g oxidant-to-apolipoprotein molar ratios revealed a concentration-dependent modification of apo A-I: at a low molar oxidant-to-lipoprotein ratio (5:1) the peaks corresponding to the methionine-containing tryptic peptides T11 (residues 84-88), T16 (residues 108-116) and T22 (residues 141-149), locate d in the central region of apo A-I, disappeared. Their loss was accompanied by the formation of three oxidation products with a molecular mass 16 a,m. u. higher than that of the native peptides. This indicates the addition of oxygen, most probably caused by the oxidation of Met(86), Met(112) and Met( 148) to the corresponding methionine sulphoxides. At a molar NaOCl-to-apo A -I ratio of 10:1 the disappearance of peptides TI (residues 1-10), T7 (resi dues 46-59) and T9 (residues 62-77) was accompanied by the occurrence of ne w peaks 33.5 and 33.1 a.m.u. higher than those of the native peptides. Amin o acid analyses of peptides T7 and T9 after modification with NaOCl confirm ed that Phe(57) and Phe(71) were primary targets for oxidation by HOCl. GLC -MS analysis of hydrolysates obtained from OCl--modified T7, T9, apo A-I an d HDL, confirmed that Phe residues are an early target for OCl- modificatio n. At molar NaOCl-to-apo A-I ratios of 25:1, the peak areas of peptides T31 (residues 189-195) and T32 (residues 196-206) decreased markedly. Most imp ortantly, incubation of apo A-I with the myeloperoxidase/ H2O2/Cl- system ( the source of HOCl in vivo) resulted in almost identical modification patte rns to those observed with reagent NaOCl.