Glucose oxidase contains a disubstituted phosphorus residue. Phosphorus-31 nuclear magnetic resonance studies of the flavin and nonflavin phosphate residues.
Glucose oxidase contains a disubstituted phosphorus residue. Phosphorus-31 nuclear magnetic resonance studies of the flavin and nonflavin phosphate residues.
复制标题
葡萄糖氧化酶含有二取代的磷残基。
DOI：
10.1021/bi00506a026
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Husain,M
中科院分区：
文献类型：
--
作者：
James,TL;Edmondson,DE;Husain,M
埃尔克哈特，In.该酶制剂经超滤浓缩后，用Sephadex G-100在0.1M三乙酸乙酯、pH 8.0的条件下进行层析。所用各种酶制剂的不同批号在NaDodS04凝胶电泳(Weber&Osborne，1969)上显示单一条带，并显示相同的磷/黄素比率以及碳水化合物含量。按照Swoboda&Massey(1966)的描述，制备了脱辅酶和原酶降解形式的脱辅酶。碳水化合物分析用Orci-nol-硫酸方法(Ashwell，1957)，蛋白质浓度用缩二脲方法(Gornall et al.，1949)估计，磷浓度用Bartlett(1959)提出的方法测定。FAD购自西格玛化工公司，未经进一步细化即可使用。在进行核磁共振光谱测量之前，所有溶液都通过在一个小的Cherex柱上的层析去除了任何受污染的顺磁性杂质。31P核磁共振谱在Varían XL-100-15型光谱仪上于40.5 MHz处获得，该光谱仪配备了Nicolet FurierTransform附件和多个其他核附件(MONA)。采用了正交位相检测，并利用样品中D20的DeuTerium共振实现了场频锁定。所有化学位移都是相对于85%磷酸的外标进行测定的。所有的光谱都是在25±3℃下获得的，样品在12 mm的精密核磁共振管(Wilmad 514A-7PP)中获得。用厌氧核磁共振管(Wilmad 514A-7-SJ)，在无氧Ar气氛下，获得了葡萄糖氧化酶的半喹酮和完全还原形式的光谱。所有的实验都是用不同的样品进行的一式两份或三份。
Elkhart, IN. After concentration by ultrafiltration, the enzyme preparation was further treated by chromatography on Sephadex G-100 in 0.1 M Tris-acetate, pH 8.0. The different lot numbers for the various enzyme preparations used revealed a single band on NaDodS04 gel electrophoresis (Weber & Osborne, 1969) and showed identical phosphorus to flavin ratios, as well as carbohydrate content. The apoenzyme and Pronase-degraded forms of the apoenzyme were prepared as described by Swoboda & Massey (1966). Carbohydrate analyses were performed by the orci-nol-sulfuric acid method (Ashwell, 1957), protein concen-trations were estimated by the biuret method (Gornall et al., 1949), and phosphorus concentrations were determined by the procedure outlined by Bartlett (1959). FAD was purchased from the Sigma Chemical Co. and used without further pu-rification. Prior to NMR spectral measurements, all solutions were freed of any contaminating paramagnetic impurities by chromatography on a small Chelex column. 31P NMR spectra were obtained at 40.5 MHz on a Varían XL-100-15 spectrometer equipped with a Nicolet Fouriertransform accessory and a multiple othernuclei accessory (MONA). Quadrature phase detection was employed and field-frequency locking was implemented by using the deu-terium resonance of the D20 inthe sample. All chemical shifts were determined relative to an external standard of 85% phosphoric acid. All spectra were obtained at 25±3 C with the samples in 12-mm precision NMR tubes (Wilmad 514A-7PP). Spectra of the semiquinone and fully reduced forms of glucose oxidase were obtained by using anaerobic NMR tubes (Wilmad 514A-7-SJ) with an atmosphere of oxygen-free argon over the samples. All experiments were performed in duplicate or triplicate with different samples.