Isolation of the thiamine degradation products formed by shellfish thiaminase.

The breakdown of thiamine by the action of shellfish thiaminase was demon strated by Fujita et al. (1) to be a base exchange reaction. Thereafter, other type of thiaminase which decomposes thiamine into 2-methyl-4-amino-5-hydroxymethylpyrimidine (Pm-CH2OH) and 4-methyl-5-ƒÀ-hydroxyethylthiazole (Th) was discovered. Then, thiaminase was classified into a base exchange type (Thiaminase ‡T) and a hydrolytic type (Thiaminase ‡T‡T) (2). The nature of the base exchange reaction of Thiaminase ‡T was demonstrated by isolating heteropyrithiamine (3), quinohiamine or anilinothiamine (4) from the reaction pro ducts. However, anilinothiamine was proved to be exceptionally formed by Thiaminase ‡T‡T (1, 5), giving some confusion upon the classification. Further more, the mode of thiamine degradation by crude thiaminase ‡T preparations without adding amines remains still to be verified, because the pyrimidine derivatives formed were not detected on a paper chromatogram. On the other hand, Krampitz and Woolley (6) demonstrated that the carp thiaminase ‡T degraded thiamine into Th and Pm-CH2OH which had been se condarily formed from an unknown intermediary pyrimidine derivative and Fujita and Hasegawa (7) showed shellfish thiaminase ‡T to degrade thiamine into Pm-CH2OH and Th by isolating both compounds. Furthermore, Barnhurst and Hennessy (8) isolated ichthiamine in a crystalline form from raw Quahog clam digests, but they found indications that the yields were increased by adjusting the pH of the enzyme solution to an acid side. In order to test the physiological reaction of raw clams, the authors attemp ted to isolate all the pyrimidine derivatives supposed to be produced in the reaction with thiamine. Various isolation procedures have been attempted using an intensive absorption at 240-250mƒÊ as a criterion of the pyrimidine derivatives and it was found that the pyrimidine derivatives were completely adsorbed on a weakly acidic cation exchange resin, Amberlite IRC-50, from the reaction mixture and they can be eluted easily. By this procedure the impurities were largely removed and various pyrimidine derivatives became detectable on a paper chromatogram as Dragendorff-positive substances and showed an intensive absorption at 2537A. They could be separated from each other by using ion exchange chromatography (9). Thus, 2.0g of thiamine was degraded almost completely by shellfish thiaminase ‡T, and the pyrimidine