melitten and 2-2--azobis(2-amidinopropane)

In the present study, we examined the pattern of protein modification elicited by alkylperoxyl radicals and alkylperoxides. To this end, we exposed glutamine synthetase (GS) and the peptide melittin to solutions containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which is known to decompose in aqueous, aerobic solutions to yield alkyl radicals and alkylperoxides. Under our conditions, pH 7.4, 37 degrees C, the AAPH-dependent formation of alkylhydroperoxide increased linearly with time and led to 40% inactivation of GS in 1 h and to complete inactivation in 4 h. Complete inactivation was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyrosine residues, 5 of 16 methionine residues, and all of the tryptophan residues (2 residues) per subunit. Inactivation of GS was associated also with some protein fragmentation and the formation of some higher molecular weight aggregates. Exposure of GS to AAPH led also to the generation of protein carbonyl derivatives (0.34 mol/mol subunit) and to formation of a significant amount (0.038 mol/mol subunits) of quinoprotein derivatives. To investigate the mechanism of tryptophan modification, the 26-amino-acid peptide, melittin, which contains one tryptophan but no histidine, tyrosine, or methionine residues, was treated with AAPH. N-Formylkynurenine was identified as the major product of tryptophan oxidation in melittin.

Topics: Amidines; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glutamate-Ammonia Ligase; Histidine; Kynurenine; Melitten; Methionine; Molecular Weight; Oxidants; Oxidation-Reduction; Reactive Oxygen Species; Time Factors; Tryptophan; Tyrosine

The interactions of the lipid and protein moiety of human low-density lipoprotein (LDL) and their influence on the oxidation behavior of LDL were modified using an amphipathic peptide, melittin, as a probe. The interaction of melittin with the LDL phospholipid surface resulted in a destabilization of apolipoprotein B-100 (apoB-100) as monitored by differential scanning calorimetry, while the characteristics of lipid core melting remained nearly unchanged. Binding of melittin caused a restriction of lipid chain mobility near the glycerol backbone, but not in the middle or near the methyl terminus of the fatty acyl chains as observed by electron paramagnetic resonance. Also, upon melittin addition, the level of copper binding to apoB-100 and the oxidizability of LDL by Cu2+ ions were greatly reduced, as indicated by abolished tryptophan fluorescence quenching upon Cu2+ binding and, during oxidation, prolongation of the lag phase of oxidation, attenuated consumption of alpha-tocopherol, and a lowered maximal rate of conjugated diene formation. This reduction of oxidizability could not be reversed by increasing the Cu2+ concentration. It is deduced that interaction of Cu2+ and alpha-tocopherol is required for reductive activation of the metal. It can be abolished by interfering with the interactions between apoB-100 and the lipid moiety of LDL which modifies the conformation of LDL and, as a consequence, hinders copper binding to apoB-100.

Topics: Adult; Amidines; Apolipoprotein B-100; Apolipoproteins B; Calorimetry, Differential Scanning; Copper; Electron Spin Resonance Spectroscopy; Humans; Lipoproteins, LDL; Melitten; Oxidation-Reduction; Phospholipids; Scattering, Radiation; Spectrometry, Fluorescence; Tryptophan; X-Rays