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Studies of individual enzymes and metabolic pathways in the erythrocyte are hampered by the fact that in lysates, or even in partially purified systems, hemoglobin is present in overwhelming amounts compared to the various enzymic proteins. Although carbonic anhydrase (2), peptidase (3, 4), methemoglobin reductase (5), erythrocuprein (6), nucleoside phosphorylase (7-9), adenosine triphosphatase (10), and glutathione reductase (11) have been isolated from hemolysates, some special characteristic of the protein in each instance has made it possible to remove hemoglobin in a single operation.The present paper describes a method which can be used for the separation of many erythrocyte enzymes from hemoglobin by treatment of the hemolysate at pH 7.0 with diethylaminoethyl (DEAE) cellulose; under these conditions, proteins having an isoelectric point below 7.0 are adsorbed onto DEAE, while hemoglobin, having an isoelectric point near neutrality, is not adsorbed. DEAE cellulose has been used widely for the fractionation of proteins (12-15), and as applied specifically to red cell enzymes, Kirkman (16) has utilized this exchanger in the purification of glucose-6-phosphate dehydrogenase.The Methods. Protein was determined by the biuret method with crystalline bovine serum albumin as the standard. Hemoglobin was measured by the procedure of Evelyn and Malloy (17) and by its absorbancy at 410 mu. With the latter method, the concentration of hemoglobin was calculated, using a millimolar extinction coefficient of 120 (18). Spectrophotometric assays at a single wave length were carried out in a Beckman spectrophotometer, model DU. Absorption spectra over a large wave length region were obtained with the Beckman recording spectrophotometer, model DK-1. Inorganic and organic phosphate fractions were measured by a method described previously (19). ATP was assayed by the bioluminescent reaction of firefly extracts containing luciferin and luciferase (20,21), with the use of the G. K. Turner fluorometer. ADP was measured with the coupled reaction, pyruvic kinase-lactic dehydrogenase (22). Nucleotide profiles (23) were obtained by the ion-exchange chromatography method of Hurlbert, Schmitz, Brumm and Potter (24). 1The following abbreviations have been used: AMP, ADP, and ATP, adenosine mono-, di-, and triphosphate, respectively; DPN, and TPN, di-and triphosphopyridine nucleotide; DPNH, TPNH, reduced DPN and TPN; GTP, guanosine triphosphate; CMP, cytidine monophosphate; and UDP, uridine diphosphate. 1257

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It has been shown previously that the addition of inosine to ACD preservative 2 prolongs the effective period of in vitro storage of erythrocytes (1). Inosine is utilized by the red cell after a phosphorolytic cleavage to ribose-l-phosphate and hypoxanthine, mediated by a nucleoside phosphorylase (2, 3). Ribose-l-phosphate enters the "aerobic shunt pathway" of glucose metabolism after conversion to ribose-5-phosphate, and a subsequent effect is a generation of ATP 8 and the resultant maintenance of the energy reserve of the red cell (4). Inasmuch as the red cell lacks the enzyme, xanthine oxidase (5), it is evident that the other cleavage product, hypoxanthine, is not metabolized further but remains in the red cell and the plasma during storage.The present investigation is concerned with the rate of conversion of inosine to hypoxanthine by the red cells and the ratio of these substances in the plasma of blood stored in ACDI, as well as the in vivo metabolism of inosine after infusion. Related studies on the nucleosides, adenosine and guanosine, will be presented also. METHODSHuman blood was collected in ACD, and the nucleoside, dissolved in 0.9 per cent NaCi, was added with sterile precautions.Inosine and adenosine were obtained from Schwarz Laboratories; hypoxanthine and guanosine from Nutritional Biochemicals Corporation. adjusted to pH 10 with NH4OH, according to the methods described previously (1). After identification of the purine-containing compounds with the use of an ultraviolet light (Mineralite), the materials were eluted from the paper with water and measured spectrophotometrically. These substances were estimated also by quantitative densitometry4 of the paper chromatograms. The hypoxanthine content of the plasma and red cells was determined enzymatically, using xanthine oxidase (7), except that the assays were performed on neutralized. PCA filtrates of the various fractions.Acid filtrates of plasma were prepared in the following way: 2 ml. of plasma were added to 2 ml. of cold 0.6 N PCA, mixed well, and centrifuged at 40 C for 5 minutes at 15,200 X g.5 The precipitate was washed once, with 2 ml. of cold 0.3 N PCA, and the centrifugation was repeated. The supernatant fluid and the washing were, combined and neutralized with cold 20 per cent KOH, followed by centrifugation at 4°C for 10 minutes at 15,200 X g. The KCIO4 precipitate was washed once with 2 ml. of cold distilled water and centrifugation repeated. This second supernatant fluid and the washing were combined for analysis. Acid filtrates of the red cells were prepared in essentially the same way. Approximately 4 ml. of cells were washed twice with an equal volume of cold 0.9 per cent NaCl each time with centrifugation for 20 minutes at 1,700 X g.6 The washings were discarded, since they contained only negligible amounts of ultraviolet absorbing materials.A 50 per cent cell suspension was prepared with 0.9 per cent NaCl. Hematocrit determinations were made on this cell suspension. One ml. of cell suspension was added to 3 ml. of cold 0.6 N P...

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