Ein Verfahren zum spezifischen Nachweis kleinster Glykolylmengen in Glykoproteinen

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Abstract
The specificity of the colorimetric reaction of glycolic acid with 2,7-dihydroxy-naphthalene-sulfuric acid according to Eegriwe was tested. The homologous a-(mono)-hydroxy acids interfere to some extent, but there was a much greater interference by poly-hydroxy aldehydes and acids, especially carbohydrates, i.e., neutral sugars, amino sugars and uronic acids. The interference by these substances is due to the sometimes very broad absorption maximum that they produce at 480-500 nm, still with considerable absorption at 546 nm. Since acrolein in the Eegriwe reaction gives a maximum at 480 nm, it is assumed that under the conditions of the reaction the above poly-hydroxy aldehydes and acids are converted into unsaturated aldehydes, which react in a similar way. In order to determine the glycolyl group of N-glycolylneuraminic acid in glycoproteins, the glycolic acid must be separated from the other products of hydrolysis before it can be measured colorimetrically. These separation procedures (charcoal filtration according to Klenk and Uhlenbruck; anion exchange according to Gibbons) remove most of the above interfering substances. Some of the impurities cannot always be removed, so that it is not possible to determine glycolyl levels of less than 1% or 0.1%, respectively. For the study of the biosynthesis of the glycolyl group of N-glycolylneuraminic acid a method is required that is sensitive to less than 0.1% glycolyl in a maximal quantity of 10 mg of experimental material. In this case the material to be analyzed is hydrolyzed with acidic ethanol. The resulting ethyl glycolate is then distilled under reduced pressure to thoroughly remove it from all substances that interfere in the color reaction, and the saponified glycolate is finally measured quantitatively in the usual way with Eegriwe''s reagent. In this way, with a lower detection limit of about 0.5 ug glycolyl, 0.01% of glycolyl can still be measured optimally in not more than 5-10 mg of starting material.

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