ABSTRACT
Favourable analytical conditions allowing amino acid analysis in biological fluids, acquired from small human biopsy specimens, were achieved by considering various derivatization methods, the mode of detection and the column used. By using o-phthalaldehyde-3-mercaptopropionic acid as derivatization agent (high sensitivity and stability) and fluorescence detection (excitation at 330 nm, emission at 450 nm), excellent separation of 26 amino acids was obtained in the lower pmol range (1-10 pmol). The reproducibility of the retention times was better than 1.0% for the majority of amino acids and the results from high-performance liquid chromatography (HPLC), compared favourably with those of conventional amino acid analysis (r = 0.97). HPLC technology facilitates amino acid analysis in biopsy specimens of less than 1 mg of tissue.
Subject(s)
Amino Acids/analysis , Body Fluids/analysis , 3-Mercaptopropionic Acid , Adult , Chromatography, High Pressure Liquid , Humans , Liver/analysis , Male , Muscles/analysis , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , o-PhthalaldehydeSubject(s)
Dietary Proteins/adverse effects , Food Handling , Lysine/analogs & derivatives , Lysinoalanine/adverse effects , Animals , Chemical Phenomena , Chemistry , Cooking , Humans , Kidney/pathology , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Lysinoalanine/chemical synthesis , Lysinoalanine/toxicity , Rats , Terminology as TopicABSTRACT
Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic or non-classic routes. Some bacterial and mammalian enzymes, endo- and exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for their ability to split the epsilon-peptide bond in the above substrates. Kinetic constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas) and hog intestinal aminopeptidase were additionally examined for their Ki values with the above substrates in comparison to the classic protease substrate leucine p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be efficient in cleaving epsilon-peptide bonds.
Subject(s)
Oligopeptides/chemical synthesis , Peptide Hydrolases/metabolism , Amino Acid Sequence , Animals , Endopeptidases/metabolism , Glycine , Kidney/enzymology , Kinetics , Lens, Crystalline/enzymology , Leucyl Aminopeptidase/metabolism , Lysine , Phenylalanine , Saccharomyces cerevisiae/enzymology , Structure-Activity Relationship , Substrate Specificity , SwineABSTRACT
(Aminoacyl)n=1-10-AH-Sepharoses can easily be prepared by the N-carboxy anhydride method from amino acid N-carboxy anhydrides and AH-Sepharose 4B at pH 10.2, 0degreesC in aqueous borate buffer. Syntheses of cystinyl-, cysteinyl-, isoleucyl-, leucyl- and phenylalanyl-AH-Sepharoses are described. An improved preparation for cystine bis(N-carboxy anhydride) is presented.
