Some properties of hemoglobin A2.

While no significant physiologic function of hemoglobin A2 (Hb A2), the minor basic component of human hemoglobin, has been recognized, only its oxygen equilibria have been studied in detail. Since hemoglobin A2 and its oxidative denaturation product, hemichrome A2, bind to the red cell membrane, particularly to band 3, to a greater extent than do Hb A or hemichrome A, some of the properties of Hb A2 that might influence hemoglobin-membrane association were examined. Hemoglobin A2 exhibited slightly increased susceptibility to autoxidation to methemoglobin. No differences were noted between methemoglobins A and A2, including the rates of enzymatic reduction and stability of the heme-globin linkage. Oxyhemoglobin A2 had a slightly lower solubility in phosphate buffer than did hemoglobin A. While the hemichromes (prepared with phenylhydrazine) of hemoglobins A2 and A had the same optical spectra, the A2 hemichrome exhibited greater stability. It is suggested that the differences in products of oxidative denaturation may provide the basis for functional differences between hemoglobins A2 and A.

Growth of Plasmodium falciparum in human erythrocytes containing abnormal membrane proteins.

To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, we have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin [HS(Sp+)], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1(0))], HE due to a spectrin alpha I domain structural variant that results in increased content of spectrin dimers [HE(Sp alpha I/65)], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [3H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp+) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. Preincubation of HS(Sp+) RBCs in culture for 3 days did not alter these results. Normal parasite growth was observed in RBCs from one HS subject with normal membrane spectrin content. The extent of decreased parasite growth in HS(Sp+) RBCs closely correlated with the extent of RBC spectrin deficiency (r = 0.90). Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth inhibition, the extent of which correlated with the content of spectrin dimers (r = 0.94). RBCs from two unrelated subjects with structural variants of band 3 sustained normal parasite growth. Decreased growth in the pathologic RBCs was not the result of decreased ATP or glutathione levels or of increased RBC hemolysis. We conclude that abnormal parasite growth in these RBCs is not the consequence of metabolic or secondary defects. Instead, we suggest that a functionally and structurally normal host membrane is indispensable for parasite growth and development.

Frequencies of Band 3 variants of human red cell membranes in some different populations.

A variant of Band 3, the major protein of the erythrocyte membrane, was observed by Mueller and Morrison in 1977 in 6-7% of healthy blood donors on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes treated with pronase. Pronase treated red cells containing this first recognized variant [here designated 'Band 3-Memphis (m)'] section had two bands of about 63,000 and 60,000 Mr while pronase treated normal cells had only the lighter Mr band. The present study includes data on the frequency of variants resembling Band 3-Memphis in patients of different ethnic groups and on random donors obtained earlier in Memphis. These variants were detected by the original method of Mueller and Morrison and were not associated with recognized clinical or haematological abnormalities. Significantly higher gene frequencies for the variants of the (m) type were observed in American Indians, African Americans and Filipinos than in Caucasians; putative heterozygotes and homozygotes were identified among each of these groups. The frequency of silent Band 3 polymorphisms in different populations should be considered in the interpretation of clinical findings associated with the presence of Band 3 variants.

Reverse phase high-performance liquid chromatography and secondary ion mass spectrometry. A strategy for identification of ten human hemoglobin variants.

Ten abnormal hemoglobins were detected and characterized in individual cases referred to our laboratory for evaluation of hematological problems. Six of these variants were electrophoretically silent and could be detected by reverse phase high-performance liquid chromatography (HPLC) analysis. HPLC was also used to analyze the tryptic peptides of each individual variant. In most of these variants, secondary ion mass spectra of the mixture of the tryptic peptides could reveal the aberrant peptide and predict possible substitution through the mass difference between the normal and abnormal peptide. The mass spectra of the isolated abnormal peptide generally contained sufficient fragment ions to define the position of the amino acid substitution, obviating the need for lengthy sequencing procedures. Combination of the two techniques.

Some properties of hemoglobin mobile (alpha 2 beta 2 73 Asp----Val).

A hemoglobin variant was identified as hemoglobin Mobile in which valine replaces the normal aspartic acid at beta 73. Studies of its oxygen equilibria and of its interactions in gelation when mixed with hemoglobin S were carried out. Hemoglobin Mobile had an oxygen affinity lower than that of hemoglobin A, as observed by others. However, in mixtures with hemoglobin S, hemoglobin Mobile appeared to impair gelation or increase solubility to a slightly greater extent than did hemoglobin A. Beta 73 is a known site of intermolecular interactions in polymers of hemoglobin S. Our studies suggest that the impairment of hemoglobin S polymer formation by altered intermolecular interactions is significantly less in Hb Mobile than in Hb Korle-Bu in which beta 73 is asparagine.

Association of hemoglobin C with erythrocyte ghosts.

The interaction of hemoglobin C (Hb C) with erythrocyte membranes was studied using changes in fluorescence intensity in a membrane-embedded probe. The affinity of Hb C for the membranes at pH 6.0 and pH 6.8 was compared to that of normal hemoglobin (Hb A). Steady-state and kinetic data were delivered. The affinity of Hb C for the erythrocyte membrane at pH 6.8 appeared to be about five times greater than that of Hb A. The associations of Hb C and Hb A with the membrane were reversible to about the same extent. The cytoplasmic portions of band 3 membrane proteins were suggested to be the binding sites for both hemoglobins. The membrane binding of Hb C at pH values of 6.8 to 7.0 indicates that this reaction may occur under physiological circumstances.

Interactions of hemoglobin S with the red cell membrane.

Significant differences were observed in the binding of hemoglobin A and hemoglobin S to normal red cell membranes containing the fluorescent chromophore 12-(9-anthroyl) stearic acid. Deoxyhemoglobin S had a greater affinity for the membrane than did deoxyhemoglobin A and part of the binding of deoxyhemoglobin S appeared to be irreversible. When the hemoglobin binding site in red cell membranes, Band 3, was blocked with with glyceraldehyde-3-phosphate dehydrogenase, neither hemoglobin S nor A was bound. It is suggested that the binding sites for hemoglobin S to normal red cell membranes involves two steps: (1) the binding of hemoglobin to Band 3 in which increased binding reflects the greater positive charge followed by (2) a component of irreversible binding, believed to reflect an interaction between a hydrophobic area of hemoglobin S and red cell lipids.