Purification and properties of diphosphopyridine nucleotide diaphorase of human erythrocytes.

Diaphorase Assay-Six-hundredths pmole of 2,6-dichlorophenolindophenol, 50 pmoles of Tris-HCl, pH 7.55, 1 pmole of EDTA, enzyme, and 0.44 pmole of DPNH in a volume of 3 ml were placed in a Beckman spectrophotometer equipped with a water jacket through which water at 25” was circulated. A control cuvette without enzyme was included, and absorbancy at 600 rnp was determined for 10 minutes. Under these conditions, both the rate of the nonenzymatic reaction and that of the enzymatic reaction were very nearly first order with respect to dye concentration. Rate of reaction was defined as the apparent first order reaction constant, K, in min-1 minus the K of the control. A unit of activity was that amount of enzyme calculated to give unit value to K. Specific activity was expressed as units of activity per milligram of protein. Protein was determined by a spectrophotometric method with a correction applied for the heme protein present. Heme protein was calculated from absorption at 410 rnp with hemoglobin as a standard. The absorption at 280 rnp and 260 rnp was determined, and the absorption due to heme protein was subtracted from the total absorption at these wave lengths. Nonheme protein was then calculated by the method of Warburg and Christian (3). 2,6-Dichlorophenolindophenol (Eastman Organic Chemicals Department) was stored as a 0.0012 M solution in 0.01 M Tris-HCl, pH 8.65. Methemoglobin was a crystalline product prepared from nitrite-treated human red cells. Lipoamide was a gift of Dr. Lester J. Reed. Flavin was determined by the method of Burch, Bessey, and Lowry, (4). Pur$cution of Enzyme-Cells from outdated blood from a blood bank were washed three times with an equal volume of buffered saline solution (1 volume of 0.1 M potassium phosphate, pH 7.4, plus 9 volumes 0.9% NaCl) and frozen until ready for use. All subsequent operations were carried out in a cold (5”) room unless otherwise noted. All Tris-HCl buffers contained 1O-3 M EDTA. To 2 liters of washed cells were added 38 liters of water. The pH of the mixture was brought to 7.4 with 1 M Tris, and 11.2 kg of (NH&SO4 were added. The solution was passed through a Sharples centrifuge, and the residue in the bowl was discarded. To the supernatant fluid were added 4.2 kg of (NH&S04. After passage through the centrifuge, the supernatant fluid was discarded, and the residue was dissolved in 350 ml of 0.1 M TrisHCl, pH 7.4. This solution was centrifuged at 34,000 x g, and the precipitate was discarded. The liquid (approximately 460 ml) represented Fraction 2. To Fraction 2 were added 43 g of (NH&S04. The insoluble material was removed by centrifugation and was discarded. To the supernatant fluid were added 49 g of (NH&Sod. The precipitate was removed by centrifuging and dissolved in 50 ml of 0.1 M Tris-HCl, pH 8.65. To this solution (Fraction 3) were added 3 g of (NH&S04. The precipitate was removed by centrifuging and was discarded. To the supernatant solution were added 9 g of (NH&S04. The precipitate was collected by centrifuging, and dissolved in 10 ml of 0.1 M Tris-HCI, pH 8.65. This solution was dialyzed for 6 hours against 1 liter of 0.01 M Tris-HCI, pH 8.65, to give Fraction 4. Calcium phosphate gel on cellulose powder was prepared according to the method of Price and Greenfield (5), and a column, 10 cm in diameter, was filled to a depth of 3 cm with the gel. To this were added successively: 50 ml of 0.01 M TrisHCl, pH 8.65; the dialyzed enzyme solution; 1000 ml of 0.01 M Tris-HCl, pH 8.65; 500 ml of 0.05 M Tris-HCl, pH 8.65; and 1000 ml of 0.05 M Tris-HCl, pH 8.65, containing 1 y0 (NH&S04. This procedure was performed at room temperature. Suction was applied to the column, The first 1500 ml of eluate contained considerable protein but no enzyme activity. The final liter of eluate was collected in loo-ml fractions, and those with a specific activity greater than 1.0 were combined as Fraction 5. To Fraction 5 were added 31.5 g of (NH&S04/100 ml of eluate, and the precipitate was removed by centrifuging and was discarded. An additional 14 g of (NH&S04/100 ml of eluate were added, and the precipitate was dissolved in 2.5 ml of 0.1 M Tris-HCl, pH 8.65, to give Fraction 6. At this point, five samples of Fraction 6 were combined and dialyzed for 6 hours in 2 liters of 0.01 M Tris-HCI, pH 8.65. A Ca3(PO4)2 gel-cellulose column, 4 cm in diameter and 5 cm deep, was prepared, to which the following were added successively: 50 ml of 0.01 M Tris-HCI, pH 8.65; enzyme solution; 250 ml of 0.01 M Tris-HCl, pH 8.65; 250 ml of 0.05 M Tris-HCI, pH 8.65; and 250 ml of 0.05 M Tris-HCl, pH 8.65, containing 1% (NHMO~. The last 250 ml of eluate were collected in 25 ml portions, and those fractions with specific activity greater than 3 combined as Fraction 7. To Fraction 7 were added 32.2 g of (NH&S04/100 ml, and the precipitate was discarded. To the supernatant solution were added 12.3 g of (NH&SO&00 ml of Fraction 7. The precipitate was dissolved in 3 ml of 0.1 M Tris-HCl, pH 8.65, to give Fraction 8. Fraction 8 was dialyzed for 6 hours against 1 liter of 0.01 M