Lois R. Manning

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.

Exchange of Subunit Interfaces between Recombinant Adult and Fetal Hemoglobins EVIDENCE FOR A FUNCTIONAL INTER-RELATIONSHIP AMONG REGIONS OF THE TETRAMER

The inter-relationship between the interior subunit interfaces and the exterior diphosphoglycerate (DPG) binding region of the hemoglobin tetramer and the effects of a specific N-terminal acetylation on tetramer assembly have been evaluated. Tetrameric fetal hemoglobin F in the liganded state was found to dissociate to dimers much less than previously appreciated,i.e. about 70 times less than adult hemoglobin A (K d = 0.01 μm and 0.68 μm, for HbF and HbA, at pH 7.5, respectively) over the pH range 6.2–7.5, whereas HbF1, in which the N termini of the γ-chains are acetylated, dissociates like HbA. To determine whether this feature of HbF could be transferred to hemoglobin A, the single amino acid difference in their α1β2/α1γ2interfaces and the 4 amino acid differences in their α1β1/α1γ1interfaces have been substituted in HbA to those in HbF. This pentasubstituted recombinant HbA/F had the correct molecular weight as determined by mass spectrometry, the expected mobility on isoelectric focusing, the calculated amino acid composition, and normal circular dichroism properties, oxygen binding, and cooperativity. Although HbA/F has the same amino acid side chains that bind DPG as HbA, its diminished response to 2,3-DPG resembled that of HbF. However, its tetramer-dimer dissociation constant (K d = 0.14 μm) was between that of HbA and HbF despite the fact that it was composed entirely of HbF subunit interfaces. The results indicate that regions of the tetramer distant from the tetramer-dimer interface influence its dissociation and, reciprocally, that the interfaces affect regions involved in the binding of allosteric regulators, suggesting flexible long range inter-relationships in hemoglobin.

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.

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.

Enzymic cleavage of the blocked amino terminal residues of peptides.

The substrate specificity of an enzyme that removes some N-terminal blocked amino acids from blocked peptides has been further explored with several naturally-occurring peptides. Chloride ion is an effective modulator of enzyme activity. Although the relative efficiency of the enzyme varies considerably with different peptide substrates, in each case there was significant although less than quantitative release of the N-terminal blocked amino acid. The possible application of this enzyme to structural studies on polypeptides is evaluated.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer–dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic ζ‐subunits leads to an average 20‐fold weakening of tetramer–dimer interfaces compared to corresponding hemoglobins containing adult α‐subunits. The dimer–monomer interfaces of these hemoglobins differ by at least 500‐fold in their strengths; such interfaces are weak if they contain ζ‐subunits and exchange with added β‐subunits in the form of β4 (HbH) significantly faster than do those with α‐subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland‐2 (ζ2β2) < Hb Portland‐1 (ζ2γ2) ≅ Hb Gower‐1 (ζ2ε2) < Hb Gower‐2 (α2ε2) < HbF1 < HbF (α2γ2) < HbA2 (α2δ2), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer–monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic → fetal → adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

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.

N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity.

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N‐terminals of either the α‐ or the β‐subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N‐terminals of the α‐subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer–dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N‐terminals of the β‐subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer–dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the α‐subunit, the slope of the plot of the tetramer–dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the α‐subunits is less extensive than that of the β‐subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

Expression and Properties of Recombinant HbA2 (α2δ2) and Hybrids Containing δ-β Sequences

Hemoglobin A2 (α2δ2), which is present at low concentration (1–2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only α and δ genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant α2δ2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the α + β or the α + γ genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.