Methods

Crystallization Procedures-Most of the preparations from human adult blood were made from cells recently separated by cent, rifugation from fresh titrated specimens, containing about 0.5 per cent of sodium citrate dihydrate (75 ml. of a 3.2 per cent solution of the salt, slightly more than isotonic, per approximately 400 ml. of blood). The essential point is the securing of solutions of hemoglobin of suitable concentration. The corpuscles are t, horoughly washed and packed by successive mixing and centrifuging, one time with 0.9 per cent WaCl and three times with a mixture of 1.2 per cent saline and 0.0025 M AICls. A stroma-free solution, the concentration of which in hemoglobin is of the order of 8 to 10 rnM per liter (referred to a molecular weight equivalent of 16,700), is obtained from the packed cells by dilution with 1 volume of distilled water, thorough mixing with 0.4 volume of toluene, and refrigeration overnight, followed by centrifugation and siphoning off the clear hemoglobin layer. Concentration of the hemoglobin is accomplished by dialysis in a refrigerator either against saturated (NH&S04 (700 gm. plus 1 liter of lvater, final volume of about 1390 ml.), the more commonly used procedure, or against 2.8 M phosphate buffer, pH 6.8 (3)(371 gm. of KZHP04. 3Hs0 and 160 gm. of KH2P0+ quantities approximated from the data of Green (26), made up to 1 liter), until the volume of the sac corztents is reduced by approximately/tii per cent. Under these conditions, three-fourt. hs to four-fifths of t, he sac cont. ents