Methods of assaying nonenzymatic glycosylation

Open University, Oxford Research Unit, England.
Analytical Biochemistry (Impact Factor: 2.22). 01/1989; 175(2):347-60. DOI: 10.1016/0003-2697(88)90558-1
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    • "In the TBA colorimetric method, 1-amino-1- deoxyketoses are dehydrated in boiling oxalic acid and released as 5-HMF. The sugar-free protein is removed by TCA precipitation and the 5-HMF concentration is determined colorimetrically after condensation with TBA (Furth, 1988). One mg of the glycoprotein produced about 0.2 ␮mol of 5-HMF, whereas 25 ␮mol of cellobiose (8.6 mg) produced 0.11 ␮mol of 5-HMF (data not shown). "
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    ABSTRACT: During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-mass glycoproteins that catalyze a redox reaction between O(2) and electron donors to produce hydroxyl radical. This reaction accounts for most of the hydroxyl radical produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, hydroxyl radical is believed to play a role in lignin degradation. The secreted glycoproteins also reduce Fe(III) to Fe(II) and strongly bind Fe(II). The partially purified glycoproteins contain 1-amino-1-deoxy-2-ketose (ketoamine) produced by the condensation of side-chain amino groups and carbohydrate. cDNAs and two putative genes encoding these glycoproteins, glp1 and glp2, have been isolated and sequenced. The 875bp glp1 and 864bp glp2 are found on scaffold 2 of the P. chrysosporium genome. These presumptive genes each consist of seven introns and eight exons. The latter encode a predicted protein of 138 amino acids and a 22-amino-acid signal sequence for secretion. The predicted protein sequences are nearly identical to N-terminal and internal sequences obtained from the partially purified glycoprotein. The molecular masses of the deduced mature proteins, 13,981 (glp1) and 13,970 (glp2), coincide with the molecular mass of the glycoprotein as determined by tricine-SDS-PAGE.
    Journal of Biotechnology 03/2007; 128(3):500-11. DOI:10.1016/j.jbiotec.2006.12.010 · 2.87 Impact Factor
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    • "The Maillard reaction is a complex network of reactions that occur during processing and storage of several foodstuffs. Several methods have been used for determining the extent to which it has progressed, such as the 2-thiobarbituric acid (TBA) method, periodate oxidation , borohydride reduction, and furosine and carboxymethyllysine determination (Furth, 1988). The TBA method has been widely applied in dairies. "
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    ABSTRACT: 5-(Hydroxymethyl)-2-furfuraldehyde (HMF) is formed upon heat treatment of milk and milk-resembling systems by the Maillard reaction, via its Amadori product, and also by isomerization and subsequent degradation of sugars. Traditionally, the HMF content has been used as an indicator of both degradation routes. A new analytical approach has been developed for determining the HMF formed only by the acidic degradation of Amadori products, called bound HMF, and that could be related to the extent of the Maillard reaction due to heat treatment or long-term storage of foods. Optimal conditions for the acidic digestion procedure were determined. Reversed-phase HPLC is applied for accurate measurement of bound HMF.
    Journal of Agricultural and Food Chemistry 05/1997; 45(5):1570-1573. DOI:10.1021/jf960930v · 2.91 Impact Factor
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    • "Despite the potential importance of analytical methods to detect sugar modified proteins, in solving problems in cell biology, this process remains difficult to assay. Methods for assaying glycated proteins have been reviewed recently by Furth (1988). The most specific and widely used assay is the furosine method, where glycated proteins are hydrolyzed in 6~ HCL for 18 h and the hydrolysates are analyzed by HPLC for the presence of furosine (E-N-(2-furoylmethyl)-L-lysine), a specific cyclization product of lysine AP. "
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    ABSTRACT: The fluorescence resulting from the reaction of a protein with fluorescamine is an indication of the number of free amino groups in that protein, consequently glycated proteins which have fewer free amino groups will show less fluorescence. With the appropriate standards, the difference in fluorescence of glycated and non-glycated proteins can be translated into the number of glycated sites in a protein. Glucose (0·22 m final concentration) was incubated with bovine serum albumin (BSA) (0·1 mg/ml) in a total volume of 50 ml of 0·1 m phosphate buffer pH 7·2 at 37°C for a period of 35 days. In order to assess the effect of metal catalyzed free radical damage to the protein, the incubation was also carried out in the presence of a metal chelating agent EGTA (0·38 mg/ml). As expected, the fluorescence of both mixtures decreased with time, as more lysyl groups became glycated. Calculations based on the standard curve of t-BOC-lysine showed that approximately 18 mol of glucose were incorporated per mol of BSA during the incubation in the presence of EGTA and 23 mol in the absence of EGTA. This difference is explained by the metal catalyzed free radical fragmentation of BSA which exposes more N-terminal amino groups for reaction with glucose.
    Food Research International 01/1992; 25(4):269-275. DOI:10.1016/0963-9969(92)90123-M · 2.82 Impact Factor
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