Electron Transfer in Nitrate and Sulphate Respiration 



397 



Solubilization and Purification of Particulate Nitrate Reductase 



The greater part of nitrate reductase, estimated with MVH as an electron 

 donor, of the particulate fraction was solubiHzed, when heat-treated at 60°C 

 for 5 min and kept at pH 8-3, at 4°C for 20 hr. Formate dehydrogenase and 

 cytochrome b^ were not solubilized by the procedure. Nitrate reductase thus 

 solubilized was purified to about 1 ,000 times the activity of cell paste (yield 

 10-20%) by adsorption onto and elution from calcium phosphate gel, 

 ammonium sulphate fractionation, and ultracentrifugation (1 10,000 g, 

 3-4 hr). The purified nitrate reductase with yellowish-brown colour is 

 homogeneous in ultracentrifugal and electrophoretic analysis and has a 

 specific activity as high as about 200,000 /^moles NOa" formed/mgN/hr 

 (Table 5). 



Table 5. Solubilization and purification of nitrate reductase 



It has no specific absorption peak except that of protein (275-280 m/i) 

 and the absorbance decreases gradually over the entire near-ultra-violet 

 and visible region with increasing wavelength. The difference spectrum, 

 (oxidized — reduced enzyme) showed a broad peak around 445-450 m// 

 which instantly disappeared on the addition of nitrate, with simultaneous 

 production of nitrite (Fig. 2). 



The purified enzyme preparation contains about 40 atoms of Fe per 

 molecule, on the basis of its weight of approx. 10^ g/mole, as estimated from 

 measurement of sedimentation constant, 8%^ ^ = 25-0 s, and diffusion 

 coefficient, 1)20, w = 2-27 X 10~^ cm^ sec^^, and assuming its partial specific 

 volume (Foo) as 0-75 ml/g. By emission spectrographic analysis using the 

 cathode layer method, the existence of Mo (1 atom/molecule) besides Fe was 



