Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains.

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry.

BACKGROUND: Erythrocytes contain a large amount of glutathione (GSH), which protects cells from oxidative injury. The purpose of this study was to examine whether hemoglobin (Hb) is modified with glutathione by oxidation of the thiol groups in diabetes mellitus and hyperlipidemia, and to determine the oxygen affinity of glutathionyl Hb. METHODS: Hb samples obtained from patients with type 2 diabetes, patients with hyperlipidemia, and healthy subjects were analyzed by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Glutathionyl Hb was synthesized in vitro by incubating Hb with GSH. The oxygen affinity of glutathionyl Hb was determined by measuring its oxygen dissociation curve. RESULTS: We first demonstrated that the concentration of glutathionyl Hbbeta chains is markedly increased in the diabetic patients and hyperlipidemic patients compared with healthy subjects. The in vitro synthesis of glutathionyl Hb by incubation of Hb with GSH was enhanced by adding H(2)O(2), a reactive oxygen species, into the incubation solution. The glutathionyl Hb prepared in vitro by incubating Hb with GSH showed a marked increase in oxygen affinity and a marked decrease in the Hill coefficient compared with Hb incubated without GSH. CONCLUSIONS: Glutathionyl Hb may be useful as a clinical marker of oxidative stress. The increased concentrations of glutathionyl Hb with high oxygen affinity and low cooperativity in diabetes and hyperlipidemia may lead to reduced tissue oxygen delivery.

Automatic measurement of the red cell oxygen dissociation curve identical with the whole blood curve.

Automatic measurement of the entire oxygen dissociation curve (ODC) of blood and hemoglobin provides a useful means for evaluating their gas-transport function. The automatic oxygenation apparatus previously developed by Imai et al. (1970, 1981), which uses a polarographic determination of partial pressure of oxygen and a spectrophotometric determination of oxygen saturation of hemoglobin, has mostly been used for the measurement of accurate ODCs of hemoglobin solution. However, it was not suitable for red cell suspension because a significant noise was superimposed on the absorbance signal due to light-scattering by red cells. In the present study, we have overcome this problem by using an integrating sphere for the photometric system. Through extensive tests we found the optimal experimental conditions for obtaining the red cell oxygenation data that were identical with the whole blood data with respect to the position (oxygen affinity) and shape (sigmoid character) of the ODC and its pH-dependence (the Bohr effect). The accuracy was higher than that of commercially available automatic apparatuses such as the "Hemox-Analyzer" (Technical Consulting Service) and "Hem-O-Scan" (Aminco). Thus, our method provides an easy and convenient means for obtaining accurate ODCs mimicking the whole blood ODCs from one drop of whole blood. An application of our method to the effect of blood storage on ODC is presented, demonstrating the usefulness of our method.

[Chemical evaluation and consideration on the traditional drug "ULUUS" (1)].

Traditional medicine named "ULUUS" is regarded to be the first Dutch precipation with a western name in Japan. It was found that this drug consists of Rhubarb (originated from Rheum sp.). Although it is still uncertain whether it was made only of Rhubarb by being kneaded hard into the monotonous square form, we could not find out any other ingredients except Rhubarb, on the bases of our chemical analysis.