We have recently shown that while the oxygen affinity of purified hemoglobin is very much greater than that of whole blood, the difference can be accounted for by the influence of red cell organic phosphates acting as cofactors (Benesch and Benesch, 1967;Benesch, Benesch, and Yu, 1968). Since D-2,3-diphosphoglycerate (DPG) accounts for the major portion of the phosphates in the human erythrocyte (Bishop, 1964), our studies were carried out with this compound, although some other phosphate esters, such as adenosine 5'-triphosphate (ATP), have similar effects (Benesch and Benesch, 1967).The dramatic shift of the oxygenation curve of hemoglobin toward lower oxygen affinities in the presence of DPG is caused by a reciprocal affinity of hemoglobin for DPG and oxygen. Therefore, the addition of DPG to oxygenated hemoglobin liberates oxygen and oxygenation of deoxygenated hemoglobin displaces bound DPG. This is due to the fact that DPG is bound to deoxyhemoglobin, but not to oxyhemoglobin, under physiological conditions, in a ratio of one mole of DPG per mole of hemoglobin tetramer (Benesch et al., 1968). The energy of binding accounts satisfactorily for the stabilization of the deoxy form of the protein by DPG and the consequent shift in the oxygenation curves. It is pertinent that the ratio of the molar concentration of effective organic phosphates in the human erythrocyte to that of hemoglobin is between 1.0 and 1.5 under normal conditions (Guest, 1942).We have now investigated the interaction of DPG with the isolated a and j3 chains of human hemoglobin and correlated the results with the effect of DPG on the oxygenation behavior of these subunits.Experimental.-Preparation of hemoglobin A and its subunits: Hemoglobin was prepared from freshly drawn human blood at weekly intervals as described previously (Benesch et al., 1968). The a and i chains were separated by the method of Tyuma, Benesch, and Benesch (1966) based on that of .After demercuration on thiolated Sephadex, the a chains were freed of all phosphate by rapid dialysis as described by Englander and Crowe (1965) against six changes of 500 ml 0.1 M NaCl (total dialysis time 3 hr at room temperature).For the removal of mercury from the # chains, a procedure that is more suitable for preparing larger amounts was developed; 1 M N-acetyl-DL-penicillamine (Aldrich Chemical Co., Milwaukee, Wis.) was added to a 2% solution of the mercurated protein in 0.1 M phosphate buffer, pH 7.5, to give a final concentration of 0.1 M. After 1 hr at 40C, the solution was dialyzed through two-dimensionally stretched Visking sausage casing (Craig, 1965) in the Englander apparatus against 100 vol of 0.1 M phosphate buffer, pH 7.5, for 1 hr at 40C. The treatment with N-acetyl-penicillamine followed by dialysis was repeated twice more, and finally the protein was subjected to continuous dialysis against 8-10 liters 0.1 M NaCl at the rate of 500 ml/hr. The resulting preparation was free of phosphate and methemoglobin and contained not more than 0.01 atom of mercury/chain. N-acetyl-peni...

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A beta-variant hemoglobin, first misjudged as a marked elevation of Hb A1, was found in a 68-year-old Japanese female with diabetes mellitus. This hemoglobin was isolated by Bio-Rex 70 chromatography combined with chromatofocusing, and was found to be Hb Hope, beta 136(H14)Gly----Asp, by classical and high performance liquid chromatographic peptide mapping techniques. Intrinsic oxygen affinity of this hemoglobin was approximately one-third as compared with that of Hb A0. This property was still observed in the constituent beta subunits isolated. Effects of such allosteric effectors as H+ (at a fixed concentration of Cl-), anion (Cl-), 2,3-diphosphoglycerate and carbon dioxide were more or less depressed. Among others, a marked reduction in the carbamate effect should be noted in a structural interpretation of the functional modifications. Subunit cooperativity, on the contrary, was not different from that in Hb A0 (n = 2.8-2.9). Explanation of these altered functions were attempted on the basis of the altered structure. The reduced stability of Hb Hope is also described.

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The reducing reaction of 2,6-dichlorophenolindophenol by L-ascorbic acid was used to determine the dead time of a stopped-flow instrument. Because this reaction is irreversible, the dead time could be determined by a simple graphical analysis. The dead time values determined by the present method were comparable to those by other methods previously reported.

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for human erythropoietin (EPO) using two anti-EPO monoclonal antibodies has been compared with an in-vitro EPO bioassay based on CFU-E colony formation in fetal mouse liver cell cultures. In normal subjects and non-uraemic anaemic patients the plasma EPO values estimated with the ELISA correlated well with those by the bioassay, and also inversely with the values of blood Hb, PCV and RBC counts. Dose-response curves for plasma and standard recombinant human EPO in the ELISA were parallel to each other. These results further confirm the validity for the ELISA in measuring circulating human EPO.

In an attempt to assess the erythropoietin (Epo) production site(s) in rat kidney, Epo response to hypoxia and renal histopathological changes were studied in rats administered with graded doses of gentamicin. Male Sprague-Dawley rats of 9 to 11 weeks old were used. Following a 14-day subcutaneous administration (67.5 or 33.8 mg kg-1 day-1) of gentamicin, a nephrotoxic aminoglycoside, selective proximal tubular lesions were produced. These gentamicin-administered rats were compared with normal rats with respect to Epo response to hypoxia. Two different kinds of hypoxic load, either 0.35 atm hypobaric hypoxia (PIO2 = 46 torr) or acute anaemia (Ht: 29.3 +/- 0.2% and [Hb]: 9.7 +/- 0.3 g dl-1) by withdrawing of blood corresponding to 1-2% of body weight was used. During the hypoxic period of up to 48 h, the peak renal venous plasma Epo titres of 3.1 +/- 0.6 and 4.3 +/- 0.6 U ml-1 was observed at the 6th h in normal hypobaric hypoxic and anaemic rats, compared with the prehypoxic value of 0.7 +/- 0.1 U ml-1. The Epo titres then declined gradually. In the rats which were administered gentamicin, Epo response pattern was the same as that observed in the normal rats, but the peak value decreased significantly to 0.8 +/- 0.3 and 1.1 +/- 0.4 U ml-1 in hypoxic and anaemic rats (P < 0.05). Histological examination revealed the selective damage to renal proximal convoluted tubules. The Epo response was reduced by the tissue damages, and restoration of the gentamicin-induced tissue injury was accompanied with restored Epo response to hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

We devised a rapid and simple method to obtain oxygen dissociation curve (ODC) for a small amount of blood with simple equipment. This system achieved the gas-blood equilibrium within 3 min. Oxygen saturation of the equilibrated blood was measured spectrophotometrically while the pH, Pco2, and Pot was measured with a Radiometer blood gas analyzer system. Whole procedures to construct ODC from the 6 point measurements could be performed within 1 hr. The standard ODC for 25 normal Japanese adults showed a mean P50 of 27.6±1.1 Torr (pH 7.40, Pco2 40 Torr, 37°C), a slightly higher value than previously reported. The discrepancy, however, can be eliminated when corrected for a slightly lower S02 by the present procedure. The standard curves for the adult blood at pH 7.20 and 7.60 and for the cord blood at pH 7.40 were also described. The "single point" procedure, a much quicker approach to measure the P50 (ABERMAN et al., 1975), was scrutinized by comparing with the "whole curve" method. The P50's by the two methods were not significantly different, mean±S.D. of the differences being 0.4±2.5 Torr (n=126) for the adult blood at pH 7.40, Pco2 40 Torr and 37°C. Similar results were obtained for the adult blood at pH 7.20 and 7.60 and for the cord blood at 7.40. We concluded that the "single point" method was sufficiently accurate and reliable.