James M. Manning

The Effects of Cyanate In Vitro on Red Blood Cell Metabolism and Function in Sickle Cell Anemia

Cyanate, which is in equilibrium with urea, combines with the alpha-amino group of the aminoterminal valine of hemoglobin in an irreversible, specific carbamylation reaction. Partial carbamylation (0.72 residues/hemoglobin tetramer) as determined by cyanate-(14)C incorporation or hydantoin analysis diminishes the in vitro sickling phenomenon. Since cyanate may react not only with hemoglobin but also with functional groups of other red blood cell proteins, the in vitro effect of cyanate was studied on sickle cells. Cells were incubated with 10 mM KCl (control) or 10 mM KNCO (carbamylated) for 1 hr, washed, and resuspended in autologous plasma. Glycolysis, ATP and 2,3-diphosphoglyceric acid (DPG) stability, autohemolysis, and osmotic fragility were not affected by carbamylation. Potassium loss in carbamylated cells (2.8 mmol/liter) was less than in control cells (9.0 mmol/liter). Pyruvate kinase activity of carbamylated cells was decreased ( approximately 25%) but the activities of other glycolytic enzymes were similar to those of control cells. Oxygen affinity of carbamylated sickle, normal, and DPG-depleted normal cells increased, and was a sensitive index of the degree and duration of reaction with cyanate. The reactivity of carbamylated cells to DPG was similar to control cells. DPG-depleted carbamylated cells regenerated DPG and increased the P(50) when incubated with pyruvate, inosine, and phosphate. The Bohr effect of normal and of sickle cells was not affected (Deltalog P(50)/Delta pH=-0.48 and -0.53, respectively) after carbamylation. The reserve buffering capacity of plasma offset the slightly diminished ( approximately 15%) CO(2) capacity of carbamylated cells so that whole blood CO(2) capacity, pH, and P(CO2) were normal. These studies provide further support for the potential clinical use of cyanate in treating and preventing the anemia and painful crises of sickle cell disease.

Nitric oxide can modify amino acid residues in proteins

Nitric oxide derived from sodium nitroprusside binds to the heme moiety of hemoglobin and also modifies some functional groups in the protein. As hemoglobin concentration is increased, globin modification is decreased presumably due to formation of the NO complex with heme. The SH groups of hemoglobin are probably not involved in the formation of the stable product formed by NO. In the presence of inositol hexaphosphate, which binds preferentially in the cleft between the two beta-chains of hemoglobin, formation of one modified derivative was selectively reduced. With hemoglobin specifically blocked on its N-terminal residues, globin modification was also significantly reduced. Carbonic anhydrase, which is blocked at its N-terminus, was also refractory to modification. The results suggest that the N-terminal groups of some proteins can be modified by nitric oxide, perhaps by deamination.

Acylpeptide hydrolase activity from erythrocytes

Acylpeptide hydrolase, which cleaves the NH2-terminal acetylated or formylated amino acid from a blocked peptide, has been purified to apparent homogeneity from human erythrocytes. The enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. Zinc inhibits to the same extent the hydrolysis of both the most efficient and the least efficient substrates. This enzyme may play a pivotal role in the processing of polypeptide chains during biosynthesis.

Oxygenation in tumors by modified hemoglobins

The effect of systemic injection of modified hemoglobin (Hb) prepared from bovine. human, or mouse Hb on tumor oxygenation was investigated. Hb was modified by (1) diisothiocyanatobenzenesulfonate (DIBS) to yield cross‐linking within a tetramer; (2) glycolaldehyde (Glyal) to yield cross‐linking between and within tetramers; (3) carboxymethylation (Cm) to change oxygen affinity: or (4) poly(ethylene glycol) (PEG) to yield attachment between tetramers. HGL9 (human glioma) in nude mice and FSaII (mice fibrosarcoma) in C3H mice were used as tumor models. Dose and time dependency were detected in the oxygenation effect by bovine‐PEG‐Hb. Internal cross‐linkage prolonged the half‐life in the circulation, and thus showed a significant effect. Compared to bovine‐CmHb, bovine‐DIBS‐Hb and bovine‐DIBS‐CmHb were more effective. Decreasing the oxygen affinity by Cm significantly enhanced tumor oxygenation. Human‐DIBS‐CmHb was more effective than human‐DIBS‐Hb. These effects were caused by oxygen carrying capacity of modified Hbs as well as hemodynamic factors, and the injection seemed to reduce both perfusion‐limited (acute) and diffusion‐limited (chronic) hypoxia.

The functional, oxygen-linked chloride binding sites of hemoglobin are contiguous within a channel in the central cavity.

Chloride ion is a major allosteric regulator for many hemoglobins and particularly for bovine hemoglobin. A site-directed reagent for amino groups, methyl acetyl phosphate, when used forglobal rather thanselective modification of R (oxy) and T (deoxy) state bovine hemoglobin, can acetylate those functional amino groups involved in binding of chloride; the extensively acetylated hemoglobin tetramer retains nearly full cooperativity. The chloride-induced decrease in the oxygen affinity parallels the acetylation of bovine hemoglobin (i.e., their effects are mutually exclusive), suggesting that methyl acetyl phosphate is a good probe for the functional chloride binding sites in hemoglobins. Studies on theoverall alkaline Bohr effect indicates that the part of the contribution dependent on chloride and reduced by 60% after acetylation is due to amino groups, Val-1(α) and Lys-81(β); the remaining 40% is contributed by the imidazole side chain of His-146(β), which is not acetylated by methyl acetyl phosphate, and is not dependent on chloride. The five amino groups—Val-1(α), Lys-99(α), Met-1(β), Lys-81(β), and Lys-103(β)—of bovine hemoglobin that are acetylated in an oxygen-linked fashion are consideredfunctional chloride binding sites. Molecular modeling indicates that these functional chloride binding sites are contiguous from one end of the central cavity of hemoglobin to the other; some of them are aligned within a chloride channel connecting each end of the dyad axis. A generalization that can be made about hemoglobin function from these studies is that the blocking of positive charges within this channel either by binding of chloride or other anions, by covalent chemical modification such as acetylation, or by site-specific mutagenesis to create additional chloride binding sites each accomplish the same function of lowering the oxygen affinity of hemoglobin.

Normal and Abnormal Protein Subunit Interactions in Hemoglobins

The characteristic properties of hemoglobin are due to the manner in which its individual subunits bond to one another first as an ab dimer and then as an a2b2 tetramer. These subunit interactions also control the binding of allosteric regulatory molecules because of sites they create as they interact with one another. Some of these interactions in hemoglobin change in the transition between its tetrameric oxy (R, for “relaxed”) or deoxy (T, for “tense”) conformational states; adult human hemoglobin A (a2b2) functions as the physiological carrier of O2 between the arterial and the venous circulation in these two conformations, respectively. The transition between these quaternary states is accompanied by concerted changes in the tertiary structure of the individual subunits upon O2 binding known as cooperativity, which is responsible for the sigmoidal shape of the O2 equilibrium curve (1–3). Myoglobin delivers O2 during muscle contraction, as described in a recent minireview (4), and it has a hyperbolic O2 equilibrium profile, i.e. no cooperative interactions because it is a single subunit protein. In tetrameric hemoglobin certain sites between the subunits at the quaternary level have the precise geometry or chemical reactivity to bind 2,3-diphosphoglycerate (2,3-DPG), protons, and chloride preferentially to the deoxy conformational state and hence shift the equilibrium away from the oxy conformation, thereby favoring O2 release. In each quaternary tetramer the oxy and deoxy dimer pairs interact differently to form the two types of tetramer-dimer interfaces in the R and T states. The strength of these interactions influences O2 binding or release in these respective states and determines how easily the tetramer dissociates to dimers. In human Hb, dimers themselves are held together by strong interactions between their aand b-subunits that do not differ significantly for the two R and T conformations.

The acetylation state of human fetal hemoglobin modulates the strength of its subunit interactions: long-range effects and implications for histone interactions in the nucleosome.

The source of the 70-fold increased tetramer strength of liganded fetal hemoglobin relative to that of adult hemoglobin between pH 6.0 and 7.5 reported earlier [Dumoulin et al. (1997) J. Biol. Chem. 272, 31326] has been identified as the N-terminal Gly residue of the gamma-chain, which is replaced by Val in adult hemoglobin. This was revealed by extending the study of the pH dependence of the tetramer-dimer equilibrium of these hemoglobins into the alkaline range as far as pH 9. From pH 7.5 to 9.0, the 70-fold difference in the association equilibrium constant between hemoglobins F and A lessened progressively. This behavior was attributed to the difference in the pK(a) 8.1 of Gly-1(gamma) compared to the pK(a) 7.1 value of Val-1(beta) of hemoglobins F and A, respectively. Evidence for this conclusion was obtained by demonstrating that natural hemoglobin F(1), which is specifically acetylated at Gly-1(gamma) and hence unable to be protonated, behaves like HbA and not HbF in its tetramer-dimer association properties over the pH range studied. An increased degree of protonation of the gamma-chain N-terminus of hemoglobin F from pH 9.0 to 8.0 is therefore suggested as responsible for its increased tetramer strength representing an example of transmission of a signal from its positively charged N-terminal tail to the distant subunit allosteric interface where the equilibrium constant is measured. An analogy is made between the effects of acetylation of the fetal hemoglobin tetramer on the strength of its subunit interactions and acetylation of some internal Lys residues within the N-terminal segments of the histone octamer around which DNA is wrapped in the nucleosome.

Site-specific modification of hemoglobin by methyl acetyl phosphate

Methyl acetyl phosphate, which was originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase [R. Kluger and W.-C. Tsui (1980) J. Org. Chem. 45, 2723], also has an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin. Three residues in or near this cleft between the beta-chains are acetylated by this reagent, i.e., Val-1, Lys-82, and Lys-144. There is no detectable acetylation of any of the amino groups of the alpha-chain. These results indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin.

Studies on the Amadori rearrangement in a model system: Chromatographic isolation of intermediates and product

The pH profile of the reaction of glyceraldehyde with either valylhistidine or alanylhistidine exhibits an optimum near pH 6.5. One of the intermediates in the reaction, the Schiff base (aldimine), can be readily detected on an amino acid analyzer. The product of the reaction, the ketoamine formed after Amadori rearrangement of the aldimine, has been isolated by chromatography on Dowex 50. Its structure has been established by elemental analysis, amino acid analysis, and the relative amounts of carbonyl and histidine moieties. These chromatographic systems should facilitate studies on the mechanism of this reaction as it relates to peptides and proteins.

Effects of Glyceraldehyde on the Structural and Functional Properties of Sickle Erythrocytes

The d- and l-isomers of glyceraldehyde are equally effective in the inhibition of SS erythrocyte sickling in vitro. The following compounds at a concentration of 20 mM were ineffective in inhibiting sickling: glyceraldehyde-3-phosphate, d-erythrose, d-ribose, d-fructose, d-glucose, d-sucrose, dihydroxyacetone, and methylglyoxal. Glyceraldehyde does not reverse the sickling of cells in the deoxy state. The properties of purified hemoglobin after treatment with glyceraldehyde and of the hemoglobin isolated from treated cells are very similar; these results suggest that glyceraldehyde itself is the reactive species within the erythrocyte. Erythrocyte glutathione is decreased by treatment in vitro with the aldehyde. Relatively high concentrations of glyceraldehyde (50 mM) lead to a small amount (3%) of cross-linking between hemoglobin monomers as well as to some cross-linking of erythrocyte membrane proteins. Lower concentrations of dl-glyceraldehyde (5-20 mM), which reduce the sickling of erythrocytes in vitro, lead to proportionally less cross-linking of hemoglobin. Cells that have been treated with those concentrations of the aldehyde show little change in their osmotic fragility, exhibit improved filtration properties, and have a lowered viscosity.