Generation of enantiomeric amino acids during acid hydrolysis of peptides detected by the liquid chromatography/tandem mass spectroscopy.
ABSTRACT The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.
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ABSTRACT: Ovalbumin, a non-inhibitory member of serine proteinase inhibitors (serpin), is transformed into a heat-stabilized form, S-ovalbumin, under elevated pH conditions. The structural mechanism for the S-ovalbumin formation has long been a puzzling question in food science and serpin structural biology. On the basis of the commonly observed serpin thermostabilization by insertion of the reactive center loop into the proximal beta-sheet, the most widely accepted hypothetical model has included partial loop insertion. Here we demonstrate, for the first time, the crystal structure of S-ovalbumin at 1.9-A resolution. This structure unequivocally excludes the partial loop insertion mechanism; the overall structure, including the reactive center loop structure, is almost the same as that of native ovalbumin, except for the significant motion of the preceding loop of strand 1A away from strand 2A. The most striking finding is that Ser-164, Ser-236, and Ser-320 take the d-amino acid residue configuration. These chemical inversions can be directly related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin. As conformational changes of the side chains, significant alternations are found in the values of the chi 1 of Phe-99 and the chi 3 of Met-241. The former conformational change leads to the decreased solvent accessibility of the hydrophobic core around Phe-99, which includes Phe-180 and Phe-378, the highly conserved residues in serpin. This may give a thermodynamic advantage to the structural stability of S-ovalbumin.Journal of Biological Chemistry 10/2003; 278(37):35524-30. · 4.65 Impact Factor
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ABSTRACT: Only five mammalian species are known to be venomous, and while a large amount of research has been carried out on reptile venom, mammalian venom has been poorly studied to date. Here we describe the status of current research into the venom of the platypus, a semi-aquatic egg-laying Australian mammal, and discuss our approach to platypus venom transcriptomics. We propose that such construction and analysis of mammalian venom transcriptomes from small samples of venom gland, in tandem with proteomics studies, will allow the identification of the full range of mammalian venom components. Functional studies and pharmacological evaluation of the identified toxins will then lay the foundations for the future development of novel biomedical substances. A large range of useful molecules have already been identified in snake venom, and many of these are currently in use in human medicine. It is therefore hoped that this basic research to identify the constituents of platypus venom will eventually yield novel drugs and new targets for painkillers.Journal of Proteomics 01/2009; 72(2):155-64. · 4.09 Impact Factor
- Journal of the American Chemical Society 01/1971; 92(25):7449-54. · 10.68 Impact Factor