Flavocoenzymes are extremely versatile but the reaction catalyzed by each individual flavoenzyme is highly specific. This requires the prerequisite mechanism that controls the flavin reactivity. In order to understand this control mechanism, the author has investigated various flavoenzymes focusing on how the flavin reactivity is controlled by monitoring the electronic state of the flavin moiety and its fluctuation along the reaction pathway. In the present report, two flavoenzymes, _D-amino acid oxidase and medium-chain acyl-CoA dehydrogenase, are concerned, the major method being ^<13>C- and ^<15>N-NMR spectroscopy of these enzymes reconstituted with ^<13>C- and ^<15>N-enriched FAD. The results obtained indicate the following : 1. The reactivity of flavin in the oxidized form of the enzymes is controlled by hydrogen-bonding network in the protein matrix and by its fluctuation during the reaction sequence. 2. In the control of the flavin reactivity, the overlap between LUMO (lowest unoccupied molecular orbital) of flavin and HOMO (highest occupied molecular orbital) of the substrate plays an important role. 3. The reoxidation process of the reduced enzymes is controlled by modulation of the electron density at C (4a) of reduced flavin, this position being the site of nucleophilic attack to molecular oxygen.Flavocoenzymes are extremely versatile but the reaction catalyzed by each individual flavoenzyme is highly specific. This requires the prerequisite mechanism that controls the flavin reactivity. In order to understand this control mechanism, the author has investigated various flavoenzymes focusing on how the flavin reactivity is controlled by monitoring the electronic state of the flavin moiety and its fluctuation along the reaction pathway. In the present report, two flavoenzymes, _D-amino acid oxidase and medium-chain acyl-CoA dehydrogenase, are concerned, the major method being ^<13>C- and ^<15>N-NMR spectroscopy of these enzymes reconstituted with ^<13>C- and ^<15>N-enriched FAD. The results obtained indicate the following : 1. The reactivity of flavin in the oxidized form of the enzymes is controlled by hydrogen-bonding network in the protein matrix and by its fluctuation during the reaction sequence. 2. In the control of the flavin reactivity, the overlap between LUMO (lowest unoccupied molecular orbital) of flavin and HOMO (highest occupied molecular orbital) of the substrate plays an important role. 3. The reoxidation process of the reduced enzymes is controlled by modulation of the electron density at C (4a) of reduced flavin, this position being the site of nucleophilic attack to molecular oxygen.

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A new sensitive and highly reactive fluorescence-labeling reagent (DMEQ-TAD) targeting conjugated dienes was developed. The fluorescent dienophile (DMEQ-TAD), in which fluorescent dimethoxyquinoxalinone (DMEQ) group is substituted via ethylene spacer on a dienophile, 1,2,4-triazoline-3,5-dione (TAD), was synthesized in 8 steps in 24% overall yield from dinitroveratrole. The reactions of DMEQ-TAD with six major vitamin D metabolites and some synthetic analogs were examined under various conditions. The reaction produced the corresponding 6,19-cycloadduct as a pair of the C (6) epimers (8 and 9) in quantitative yield. The structures of the adducts (8 and 9) including the stereochemistry at C (6) were unambiguously determined. The fluorescence-labeled vitamins were analyzed by HPLC with fluorescence detector. The new fluorometric method was used in the assay of plasma 25-OH-D_3, 24,25 (OH)_2D_3, and 25,26 (OH)_2D_3. The method was proved to be precise and reliable by comparing with the HPLC-UV method. The reaction of DMEQ-TAD with vitamin A metabolites and fluorometric assay of plasma retinoic acid was also examined.

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ラットに18%ダイズタンパク質 B_<12> 欠乏飼料を給与,pair-feeding で90日間飼育し,血漿タンパク質レベル,肝臓 xanthine oxidase 活性,尿中窒素化合物排泄量を測定, B_<12> 欠乏ラットの飼料タンパク質の利用を調べた.B_<12> 欠乏ラット尿中 MMA 排泄量は顕著な増加を示し,肝臓 B_<12> レベルの著減から,ラットの B_<12> 欠乏状態が評価された.B_<12> 欠乏ラットの成長は pair-feeding, ad libitum-feeding の対照ラットに比べ有意な遅延が認められた.血漿総タンパク質,アルブミンレベルは B_<12> 欠乏により pair-feeding の対照に比べ低下したが,尿素窒素レベルは増加を示した.また B_<12> 欠乏ラットの肝臓 xanthine oxidase 活性は低下した.次に尿中質素化合物排泄量は尿素窒素,アラントインおよびクレアチニンいずれも B_<12> 欠乏により pair-feeding の対照に比べ低下することが認められた.以上の結果から,明確な B_<12> 欠乏ラットでは飼料タンパク質の利用の低下が示唆された.また血漿および尿中尿素窒素の変動は飼料タンパク質の利用低下に対する適応と推察された.

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