A reaction for the simple sensitive fluorimetric assay of heparin and 2-amino sugars

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Abstract
3,5-Diaminobenzoic acid reacted rapidly with the product from HNO2 deamination of heparin, heparan sulphate and 2-amino-2-deoxyhexoses under very mild conditions (pH 3.0 and 37.degree. C) to give stable fluorescent derivatives. The fluorescence yield was rectilinearly related to the concentration of heparin etc. Less than 0.1 .mu.g of 2-amino-2-deoxyhexose was easily measurable in standard cuvettes. The deamination products of glucosamine and particularly galactosamine were labile in the HNO2 reagent, with half-lives of 20-40 min at room temperature. At 0.degree. C they were much more stable. The analogous product from heparin was not so labile. Under the standard conditions and at room temperature, relative fluorescence yields (D-glucosamine = 1.0) were: D-galactosamine, 0.75; D-gulosamine, 0.38; D-mannosamine, about 0.20. Neutral sugars, chondroitin sulfates, DNA and N-acetylneuraminic acids did not react, nor did N-acetylamino sugars or non-deaminated hexosamines. The Dische-Borenfreund indole method, the Kissane-Robins DNA assay and the proposed amino sugar method are all examples of simple aldehyde reactions. The specificity of the proposed method is considerably greater than the Dische-Borenfreund procedure, partly because of the much milder reaction conditions. The proposed method is very reproducible, .apprx. 50-100 times as sensitive as the Elson-Morgan reaction and 10-50 times as sensitive as the Dische-Borenfreund procedures. It is also convenient; acid hydrolysates of amino sugar-containing compounds can be directly neutralized with sodium acetate solution.