- Identification of the carboxymethyllysine residue in the advanced stage of glycated human serum albumin
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As an advanced stage of glycation, glycated human serum albumin (G-HSA; glucose content, 2 mol of 5-hydroxymethylfurfural equivalent/mol of HSA) was incubated at 37°C up to 30 d in 0.2 M phosphate buffer, pH 7.4, with 100 μM Fe3+. G-HSA incubated for 30 d (G-HSA-30(Fe)) was subsequently hydrolyzed at 110 °C for 24 h in 6 N HCl. In the hydrolysate, N(ε)-carboxymethyllysine (CML) was identified by cochromatography with synthesized CML on an amino acid analyzer. pI of HSA (4.8) shifted to 4.5 in G-HSA. A more acidic fraction, pI 4.3, appeared in G-HSA-30(Fe). CML content (mol of CML/mol of HSA) of HSA and G-HSA was as follows; 0 in HSA, 0.2 in HSA-30(Fe), 0.4 in G-HSA and 1.5 in G-HSA-30(Fe) pI 4.3. The amino acid compositions also changed in lysine, arginine and tyrosine at the advanced stage of the reaction.
- Takanashi,Sakurai,Tsuchiya
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- Mechanism of aqueous decomposition of trichloroethylene oxide
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The aqueous decomposition of trichloroethylene (TCE) oxide is shown to involve both pH-independent and hydronium ion-dependent regions. C-C bond scission is a major reaction at all pH values. Disappearance of TCE oxide is the rate-determining step for the formation of CO under the conditions studied. The product distribution of CO and three carboxylic acids (HCO2H, Cl2CHCO2H, and glyoxylic acid) did not change considerably over the pH range of -1.5-14, in general, even though the hydrolysis mechanism changes from hydronium ion-dependent to pH-independent. Mechanisms for the hydronium ion-dependent and pH-independent hydrolysis of TCE oxide were elucidated on the basis of the results of H218O and H incorporation and identification of products of the reaction of TCE oxide with lysine in both H216O and H218O. In the pH-independent hydrolysis, a zwitterionic intermediate could be formed and undergo an intramolecular rearrangement (Cl- shift) to generate dichloroacetyl chloride, which would subsequently decompose to Cl2CHCO2H. The zwitterionic intermediate could also hydrolyze at the less sterically hindered methylene to give a glycol anion, which would dehydrohalogenate to form an oxoacetyl chloride intermediate. The oxoacetyl chloride could hydrolyze to generate either glyoxylic acid, as a final product, or an anionic intermediate, which could go through a concerted mechanism to generate CO, HCO2H, and chloride. A mechanism proposed for the hydronium ion-dependent hydrolysis is very similar to that for the pH-independent hydrolysis except for the first step, which involves hydronium ion attack on TCE oxide to form a TCE-oxide cation intermediate. The lysine amide adducts were characterized by HPLC and mass spectrometry as those resulting from reaction with the postulated acyl chlorides.
- Cai, Hongliang,Guengerich, F. Peter
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- Determination of Furosine as a Measure for Irreversibly Bound Glucose in Human Fibrinogen
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An analytical procedure is presented which enables for the determination of irreversibly, nonenzymatic glycation of fibrinogen isolated from human blood.The method is based on determination of furosine, which is formed during hydrolysis of Amadori-products.For validation of the worked out furosine assay, synthesis of Nα-formyl-Nε-(desoxy-1-D-fructosyl-1)-L-lysine as a model substance and furosine as a calibration standard were necessary.Several hydrolysis experiments showed the extent of furosine formation from fructosyllysine and from human fibrinogen.The increase of fibrinogen glycation by incubation with D-glucose could also be confirmed according to the literature.Using well-known techniques for sampling, work up and in performing reduction and hydrolysis steps, quantitative determination of furosine by high-performance liquid chromatography is possible.By application of the analytical assay, the extent of glycation of human fibrinogen can be analyzed with good precision.Calibration is performed by means of a prepared furosine standard.The practical handling of the method is shown. - Keywords.Human fibrinogen; Furosine; Glycation; HPLC; Nα-Formyl-Nε-(desoxy-1-D-fructosyl-1)-L-lysine.
- Szoelgyenyi, Gerald P.,Winsauer, Karl J. B.,Deutsch, Erwin
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