Edmondson D E, Kenney W C, Singer T P
Biochemistry. 1976 Jul 13;15(14):2937-45. doi: 10.1021/bi00659a001.
In addition to 8alpha-(N3-histidyl)riboflavin, 8alpha-(N1-histidyl)riboflavin is also formed during the reaction of Nalpha-blocked histidine with 8alpha-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8alpha-(N1-histidyl)riboflavin are inditical with those of the histidylflavin isolated from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase but differ from those of 8alpha-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8alpha substitution is shown by the electron paramagnetic resonance and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the proton magnetic resonance spectrum of the oxidized form. The site of histidine substitution by the 8alpha-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150 degrees C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylflavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer has been identified as riboflavin. The compound obtained on acid treatment of 8alpha-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in thin-layer chromatography, but not in absorption, fluorescence, and electron paramagnetic resonance spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the proton magnetic resonance spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8alpha-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase shows that both isomeric forms of 8alpha-histidylflavin occur in nature.
除了8α-(N³-组氨酰基)核黄素外，在Nα-封闭的组氨酸与8α-溴代四乙酰核黄素的反应过程中还生成了8α-(N¹-组氨酰基)核黄素，其产量占组氨酰基黄素组分总量的20 - 25%。8α-(N¹-组氨酰基)核黄素的性质与从硫胺素脱氢酶和β-环匹阿尼酸氧化环化酶中分离得到的组氨酰基黄素相同，但与8α-(N³-组氨酰基)核黄素不同。这些性质包括荧光猝灭的pKa、pH 5.0时的电泳迁移率、储存稳定性以及被硼氢化钠还原的性质。阳离子半醌形式的电子顺磁共振和电子-核双共振光谱，以及氧化形式的质子磁共振光谱都证明了8α位的取代。通过用碘甲烷对咪唑环进行甲基化、在150℃下酸水解甲基组氨酸-黄素键以及通过电泳鉴定甲基组氨酸异构体，表明了黄素部分的8α-亚甲基取代组氨酸的位点。3-甲基组氨酸是N¹-组氨酰基黄素异构体的产物，而1-甲基组氨酸是N³异构体的产物。两种异构体经还原性锌裂解得到的黄素产物均已鉴定为核黄素。对8α-(N³-组氨酰基)核黄素（以前认为是N¹异构体）进行酸处理得到的化合物与母体化合物的区别仅在于核糖醇侧链，因为化学降解研究表明产物中有1-甲基组氨酸和一种黄素产物，该黄素产物与核黄素的区别仅在于薄层色谱中的迁移率，而在吸收、荧光和电子顺磁共振光谱性质方面并无差异。酸修饰仅涉及核糖醇链这一点已通过以下观察得到证明：该黄素经碱性照射产生发光黄素，该化合物的质子磁共振光谱在核糖醇质子共振区域与核黄素不同，且其高碘酸盐滴定度低于纯核黄素。8α-(N¹-组氨酰基)核黄素与硫胺素脱氢酶和β-环匹阿尼酸氧化环化酶中的组氨酰基黄素相同，这表明8α-组氨酰基黄素的两种异构体在自然界中均存在。
