Hemoglobin binds to the amino-terminal 23-residue fragment of human erythrocyte band 3 protein

Hemoglobin, aldolase and glyceraldehyde 3-phosphate dehydrogenase are known to bind to the cytoplasmic domain of band 3 protein. Binding of glycolytic enzymes to band 3 protein is inhibited by its amino-terminal fragments. To precisely localize the sequence portion of band 3 protein to which hemoglobin binds and to see whether the same region of amino-acid sequence binds both hemoglobin and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa fragment by trypsin, cyanogen bromide and mild acid hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to hemoglobin. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1 to 23. This sequence is unusual as 14 of its residues are negatively charged, it contains no basic residues and has its amino terminus blocked. Using aldolase, glyceraldehyde-3-phosphate dehydrogenase and hemoglobin as competitive inhibitors in the binding of 23-kDa fragment, the affinity of hemoglobin to this fragment appears several-fold weaker than that of both the enzymes. These findings demonstrate that glycolytic enzymes and hemoglobin bind competitively to the same polyanionic sequence region of band 3 protein.     

Isolation and characterization of a bacterial growth-stimulating peptide from a peptic bovine hemoglobin hydrolysate

 A peptide with a bacterial-growth-stimulating activity was isolated from a bovine hemoglobin hydrolysate by reversed-phase high-performance liquid chromatography. Its primary structure and molecular mass, determined by amino acid analysis and fast-atom bombardment mass spectrometry, were identical to those of fragment 48–52 (Ser-Thr-Ala-Asp-Ala) of the β chain of bovine hemoglobin. The microbiological tests in solid media demonstrated that this peptide exhibited a growth-stimulating activity on gram-negative bacteria. Received: 18 September 1995/Received revision: 19 January 1996/Accepted: 29 January 1996     

Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.     

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane

Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.     

Bromotrifluoroacetone alkylates hemoglobin at cysteine β93

The site of alkylation of hemoglobin by bromotrifluoroacetone has been determined by peptide mapping, using a stable radioactive label for the trifluoroacetonyl function. This study and ¹H and ¹⁹F-nmr experiments confirm the site of modification to be the β93 cysteine found in the βT10 tryptic peptide fragment of the modified hemoglobin.     

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C(18) column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34-46 fragment of the alpha chain and the 34-39 fragment of the beta chain of porcine hemoglobin, with IC(50) values of 4.92 and 6.02 microM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.     

Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides

Hemoglobin is a known source of biologically active peptides with various functions. In the present study, we report for the first time the existence of natural processed hemoglobin fragments exhibiting antimicrobial activity in humans. Two antimicrobial hemoglobin-derived peptides were purified from a human placental peptide library by consecutive chromatographic steps tracking the maximum growth inhibitory activity against Escherichia coli BL21. These peptides, consisting of 17 and 36 amino acid residues, were identified as being C-terminal fragments of gamma-hemoglobin and beta-hemoglobin, respectively. The antimicrobial beta-hemoglobin fragment was also purified from lysed erythrocytes, demonstrating that proteolytic degradation of hemoglobin into small bioactive peptides already starts inside erythrocytes. The identified peptides inhibit the growth of Gram-positive and Gram-negative bacteria and yeasts in micromolar concentrations. Moreover, by LPS-binding, the beta-hemoglobin fragment reduces biological activity of endotoxins. In contrast, even at high concentrations, the identified antimicrobial hemoglobin peptides do not exhibit toxic activity on human primary blood cells. We conclude that antimicrobial hemoglobin-derived peptides could be important effectors of the innate immune response killing microbial invaders.     

Two Urinary Peptides Associated Closely With Type 2 Diabetes Mellitus

Objective

To monitor of type 2 diabetes more simply, conveniently and noninvasively, we are trying to identify the potential urinary peptides that associated with different stages of glucose control in type 2 diabetes mellitus.

Methods

Firstly, we collected urine samples from type 2 diabetic patients and normal controls. These type 2 diabetic patients were divided into two groups according to fasting plasma glucose (FPG) and hemoglobin A1c% (HbA1c), respectively. Magnetic beads based weak cation exchange chromatography (MB-WCX) was used to condense urinary peptides. The eluates were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Subsequently, ClinProt was used to profile and screen the polypeptide patterns based on different methods of grouping in diabetic patients and normal controls. Finally, the amino acid sequences of differentially expressed peptides were identified by nano-liquid chromatography-tandem mass spectrometry and the protein sources of the corresponding peptide were matched in IPI Human database.

Results

Proteomics analysis found two up-regulated peptide (m/z 2756.1 and m/z 3223.2) representations in diabetic subjects, and the two peptides increased with increases in the amount of glycosylated hemoglobin. Further, the parallelism between m/z 3223.2 and glycosylated hemoglobin was better than the parallelism between m/z 2756.1 and glycosylated hemoglobin. Area under the receiver operating characteristic of the two peptides was 0.722 and 0.661, respectively. The above-mentioned peptide m/z 2756.1 was further identified as fragment of fibrinogen alpha chain precursor and m/z 3223.2 was fragment of prothrombin precursor.

Conclusion

These results suggested the two urinary biomarkers enable monitor of type 2 diabetes patients with different stages of glucose control.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.     

Biophysical studies and anti-growth activities of a peptide, a certain analog and a fragment peptide derived from alpha-fetoprotein.

A chemically synthesized 34-amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti-growth assays were performed using the original cysteine-containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti-growth activity of the peptide, while gel-filtration experiments showed the zinc ions maintained the peptide in its anti-growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti-growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti-cancer protocols.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Heterogeneity in the globin chain composition of a human minor fetal hemoglobin component

The first hemoglobin found to contain an acetyl blocking group was the minor human fetal hemoglobin, Hb FI, present as 10-15% of the total fetal hemoglobin in umbilical cord blood red cells. Acetylation occurs at the amino-terminal glycine of the gamma-globin chain. Assays for the acetyl group by two different methods gave values less than the 2 per tetramer expected for a fully acetylated hemoglobin. We have purified acetylated fetal hemoglobin FIc to homogeneity. The globin chain composition of Hb FIc has been examined by both globin chain separation on CM-cellulose and by tryptic peptide mapping by HPLC. The identities of the gamma globin chains and of the gamma T-1 peptides were confirmed by amino acid analysis. Globin chain separation profiles showed the presence of 22.3 +/- 7.0% of gamma 0 globin (of the total gamma globin) in Hb FIc. Accordingly, the tryptic peptide maps of Hb FIc tetramers also showed the presence of a similar amount of gamma 0T-1 peptide. The gamma 0T-1 peptide was not present in the maps of isolated gamma Ic globin. It is evident that column purified Hb FIc contains a certain percentage of non-acetylated gamma-globin chains, thus indicating a hybrid globin chain composition for this minor fetal hemoglobin component.     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

Synthetic potential of Staphylococcus aureus V8-protease: an approach toward semisynthesis of covalent analogs of alpha-chain of hemoglobin S

Enzyme-catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the alpha-chain of hemoglobin S with Staphylococcus aureus V8-protease converts the chain into a fragment-complementing system by hydrolyzing the peptide bond Glu(30)-Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of alpha-chain could be achieved if conditions for the V8-protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8-protease has been now investigated by incubating V8-protease-derived fragments of alpha-chain, namely alpha 1-30 and alpha 31-47 with the enzyme at pH 6.0 in the presence of n-propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as alpha 1-47 by amino acid analysis, redigestion with V8-protease (in the absence of n-propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of alpha 1-47 is at pH 6.0, 4 degrees C, and 24 hr of incubation with 25% n-propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of alpha 1-47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8-protease and the fact that alpha 1-30 and alpha 31-141 interact to form a native-like complex, opens up an approach for the semisynthesis of covalent analogs of alpha-chain of hemoglobin S.     

Salt, phosphate and the Bohr effect at the hemoglobin beta chain C terminus studied by hydrogen exchange

Hydrogen exchange experiments using functional labeling and fragment separation methods were performed to study interactions at the C terminus of the hemoglobin beta subunit that contribute to the phosphate effect and the Bohr effect. The results show that the H-exchange behavior of several peptide NH at the beta chain C terminus is determined by a transient, concerted unfolding reaction involving five or more residues, from the C-terminal His146 beta through at least Ala142 beta, and that H-exchange rate can be used to measure the stabilization free energy of interactions, both individually and collectively, at this locus. In deoxy hemoglobin at pH 7.4 and 0 degrees C, the removal of 2,3-diphosphoglycerate (DPG) or pyrophosphate (loss of a salt to His143 beta) speeds the exchange of the beta chain C-terminal peptide NH protons by 2.5-fold (at high salt), indicating a destabilization of the C-terminal segment by 0.5 kcal of free energy. Loss of the His146 beta 1 to Asp94 beta 1 salt link speeds all these protons by 6.3-fold, indicating a bond stabilization free energy of 1.0 kcal. When both these salt links are removed together, the effect is found to be strictly additive; all the protons exchange faster by 16-fold indicating a loss of 1.5 kcal in stabilization free energy. Added salt is slightly destabilizing when DPG is present but provides some increased stability, in the 0.2 kcal range, when DPG is absent. The total allosteric stabilization energy at each beta chain C terminus in deoxy hemoglobin under these conditions is measured to be 3.8 kcal (pH 7.4, 0 degrees C, with DPG). In oxy hemoglobin at pH 7.4 and 0 degrees C, stability at the beta chain C terminus is essentially independent of salt concentration, and the NES modification, which in deoxy hemoglobin blocks the His146 beta to Asp94 beta salt link, has no destabilizing effect, either at high or low salt. These results appear to show that the His146 beta salt link, which participates importantly in the alkaline Bohr effect, does not reform to Asp94 beta or to any other salt link acceptor in a stable way in oxy hemoglobin at low or high salt conditions.     

Genetic and synteny mapping of parathyroid hormone and beta hemoglobin in cattle

Parathyroid hormone and the beta hemoglobin gene cluster, which are closely linked on human chromosome 11p15, were localized to bovine syntenic group (U7) with the gene for catalase by the use of bovine x hamster hybrid somatic cells. Restriction fragment length polymorphisms (RFLPs) were followed through informative pedigrees to determine a linkage map distance of 15.6 +/- 5.4 cM between the parathyroid hormone and hemoglobin genes. Allelic frequencies of the DNA fragment were compared in a small sampling of cattle from five different breeds.     

Spectral and oxygen-release kinetic properties of human hemoglobin bound to the cytoplasmic fragment of band 3 protein in solution

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per alpha beta dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic fragment of band 3 protein. Oxygen-release kinetic measurements also showed marked changes as a function of the concentration of the cytoplasmic fragment of band 3 protein. The higher ratio mixture had significantly different kinetic properties as compared with the lower ratio one, which in turn was different from oxyhemoglobin in solution. The significance of the formation of aggregates of band 3 protein containing oxyhemoglobin dimers is discussed in context with evidence suggesting that band 3 protein may exist as an equilibrium mixture of tetramers and dimers in the membrane.