1967 年 61 巻 5 号 p. 580-597
1. Methods for the preparation of the pure benzoate complex, holoenzyme, and apoenzyme of D-amino acid oxidase [EC 1. 4. 3. 3] are described. Chromatography with Sephadex G-75 at pH 8.3 is a satisfactory procedure for further purification of the crystalline benzoate complex prepared by the previous method. Crystalline enzyme is prepared from the gel-eluate by repeated expulsion of benzoate from the complex with an excess amount of D-alanine. The cleavage of the holoenzyme into apoenzyme and FAD is performed on a TEAE-cellulose column equilibrated at pH 5.3.
2. The physico-chemical properties of three species of the oxidase prepared by the method described above are as follows : For the benzoate complex, s020, w=6.3×10-13 sec.-1 D20, w=5.3×10-7cm.2 sec.-1; [η]=0.033 dl. g.-1;_??_=0.742ml. g.-1. For the holoenzyme, s020=7.1×10-13 sec.-1; D020, w=5.8ηl0-7 cm.2sec.-1; [η]=0.026 dl. g. 1. For the apoenzyme, s020, w=3.6×10-13 sec.-1; D20, w=6.2×10-7 cm.2 sec.-1.
From these results, the molecular weights of both the holoenzyme and the benzoate complex are calculated to be 103, 000-134, 000 and that of the apoenzyme to be about 55, 000. These results are not incon-sistent with the minimal molecular weight calculated from the amount of bound FAD, provided the holoenzyme and benzoate complex exist in a dimeric form. It is concluded that the enzyme is a basic protein bearing one molecule of FAD per 50, 000g. of protein.
3. The optical rotatory properties of the apoenzyme in M/60 pyrophosphate buffer (pH 8.3) at 25°C are found to be [α]25D=-47°; λc=236mμ; a0=-240; and b0=-72. These values indicate that it contains about 12% helices. This content is confirmed by the measurement of amplitude of the trough at 233mμ. No significant differences are found in the helical contents of the apoenzyme, the holoenzyme and its benzoate complex.
