Kinetics of subunit dissociation of partially oxygenated hemoglobin determined by haptoglobin binding

Human haptoglobin 1-1 binds very rapidly to hemoglobin dimers but not to tetramers. We have studied the binding kinetics of partially oxygenated Hb A to haptoglobin 1-1. Under the oxygenation conditions used for the measurement of the K₁ of oxygenation ($\text{Hb O}{}_2\text{Hb}\text{≤ 1\%}$, pO₂ ≤ 0.5 mm Hg), the dissociation kinetics were found to be 50 times faster than that of deoxy Hb A. This result suggested that the binding of one molecule of oxygen to hemoglobin tetramer changed the quaternary structure of the intersubunit α₁β₂ contact surface.     

Structural and functional studies of Hemoglobin Wayne: An elongated α-chain variant

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.60}$ and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ($\text{?Δ}\text{log}\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{/}\text{pH}\text{(7}\text{to}\text{8) = 0.34}$ and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l′ = 5.6 × 10⁶m^?1s^?1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l′ = 2.6 × 10⁶m^?1s^?1), whereas Hb Wayne Asp showed heterogeneous binding (l′ = 6.1 × 10⁶ and 2.6 × 10⁶m^?1s^?1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.     

The oxygen affinity of hemoglobin betaSH chains is concentration dependent.

Oxygen binding to isolated hemoglobin β^SH chains exhibits heterotropic interactions with H⁺, inositol hexaphosphate and CO₂ which implies different structures of the liganded and unliganded β chains. In order to find out if the dissociation behaviour of $\text{β}{}_4{}^{\mathrm{SH}}$ homotetramers is likewise linked to oxygenation, we have measured the oxygen affinity of the pigment as a function of the protein concentration at different pH values. We found that a decrease in protein concentration is associated with a decrease in oxygen affinity. This result accords with predictions reached from studies on the self-association of liganded and unliganded β chains. Furthermore, it was established that both at high and low protein concentrations the oxygen affinity of the β chains is pH dependent.     

Non-linear relationships between oxygen saturation and magnitude of fine structure of ultraviolet oxy versus deoxy difference spectrum in human hemoglobin

Ultraviolet difference spectra of fully oxygenated hemoglobin $\text{vs.}$ successively deoxygenated or reoxygenated hemoglobin were determined in the absence and presence of organic phosphates. Magnitude of fine structure in the difference spectrum around 290 nm, which is considered to be a partial reflection of oxygenation-induced changes in quaternary conformation of hemoglobin, was not linearly related to fractional oxygen saturation of hemoglobin of the reference cell. The non-linear feature was influenced by the organic phosphates as predicted by the allosteric model of Monod $\text{et al.}$ The present study suggests that the ultraviolet oxy $\text{vs.}$ deoxy difference spectrum measurements provide a useful way to examine the validity of the model.     

Equilibrium,kinetic and structural properties of hemoglobin Cranston,an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston³ at 20 °C in the absence of phosphate revealed a high affinity (P₅₀ = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P₅₀ = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′₄, of ～ 6 × 10⁶m^?1s^?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ～ 0·5 × 10⁶m^?1s^?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k₄～ 11 s^?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α₂β^Aβ^Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.     

Oxygen binding of the heme-containing subunits of the extracellular hemoglobin of Lumbricus, terrestris

Fully oxygenated heme-containing subunits of the extracellular hemoglobin of $\text{Lumbricus}\text{,}\text{terrestris}$ were isolated by gel filtration in 0.05 M sodium borate, pH 9.0 at 7 ± 1°C. The polypeptide chain composition of the isolated subunits was determined by SDS PAGE. Most of the subunits exhibited reversible oxygenation curves with oxygen affinities higher than the intact hemoglobin. The Hill plots were nonlinear for $\text{Lumbricus}$ hemoglobin and its subunits: the latter exhibited substantial cooperativities as evidenced by Hill constants at half-saturation in the range 2.0 to 2.8.     

Effects of prostaglandins on the affinity of hemoglobin for oxygen in human whole blood in vitro

The effect of prostaglandin on the affinity of hemoglobin for oxygen was tested on human blood $\text{in vitro}$, using six different prostaglandins at several dosage levels in fresh heparinized blood from normal donors and in stored citrated blood, and using prostaglandin E₂ on the blood from four seriously ill patients. No significant alterations in the affinity of hemoglobin for oxygen were dtected. A very small decrease in oxygen affinity in stored blood with high doses of prostaglandin was not statistically significant and would be of no physiologic significance even if real.We conclude that under the circumstances of this experiment prostaglandins do not alter the affinity of hemoglobin for oxygen in human whole blood $\text{in vitro}$.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

Oxygen equilibrium characteristics of hemoglobins of baboons, Theropithecus gelada,Papio hamadryas and Papio anubis

Hemoglobins of three baboons, Theropithecus gelada, Papio hamadryas- and Papio anubis, were purified and their oxygen equilibrium characteristics were studied. (a) Oxygen affinity, as expressed by P₅₀, oxygen partial pressure for 50% oxygen binding, was in the order of gelada hemoglobin > anubis hemoglobin > hamadryas hemoglobin although the differences were small. (b) The presence of 2,3-diphosphoglycerate reduced their oxygen affinity in a similar manner. The effect on baboon hemoglobins was greater than that on human and Japanese monkey hemoglobins. (c) The intensity of the Bohr effect, as expressed by $\text{?Δlog}\text{P}{}_{50}\text{ΔpH}$, at pH 7·4 agreed well with each other and the value was 0·62 in the presence of 2 mm diphosphoglycerate and 0·52 in its absence. These results indicate that phenotypic adaptation (acclimatory) may play an important role in the adaptation of gelada baboon to high altitudes.     

Binding of organic phosphates by human hemoglobin at alkaline pH-values

The hemoglobin-oxygen equilibrium binding curve was found to be sensitive to the addition of inositol hexaphosphate at pH 9.1. A solution of hemoglobin A in 0.050 $\text{M}$ sodium borate was half-saturated with oxygen at a partial pressure of 0.55mm Hg. Hemoglobin A in 0.050 $\text{M}$ sodium borate, 0.001 $\text{M}$ inositol hexaphosphate, pH 9.1 was half-saturated with oxygen at a partial pressure of 0.95mm Hg. The Hill plot was linear with a slope of 2.0 in the absence of phosphates. In the presence of inositol hexaphosphate the slope of the Hill plot increased from 1.0 to 2.36. The dependence of fractional saturation of hemoglobin with oxygen on concentration of inositol hexaphosphate was determined at partial pressures of oxygen of 0.46 and 1.07mm Hg.     

Kinetic origin of the pronounced Bohr effect in an annelid hemoglobin

The kinetics of oxygen and CO binding by the high molecular weight hemoglobin (ca. 3,000,000) from the annelid $\text{Cirraformia}\text{grandis}$ have been measured by stopped-flow and flash-photolysis as a function of pH. Flash-photolysis studies of the Hb1 form suggest weak heme-heme interaction for ligand binding in the ferrous state. The rate constant for oxygen dissociation increases by a factor of ca. 800 from pH 9 to pH 6. Rate constants for oxygen and CO association are virtually unchanged in the range pH 6–9.     

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features. These findings provide a new global view for allosteric interactions and invoke for a modern interpretation of the role of allosteric effectors and a reformulation of the Monod-Wyman-Changeaux model for control of allosteric systems, and other complementary models as well.     

Positive cooperativity in binding carbon monoxide to hemocyanin

The binding of carbon monoxide to hemocyanin from the crab $\text{Scylla serrata}$ has been studied by thin layer optical absorption and front face fluorescence techniques. The binding to the monomeric form is completely noncooperative whereas the binding to the native oligomeric form is found to be weakly but definitely cooperative. An analysis based on the MWC model of the oxygen and carbon monoxide binding curves indicates that the allosteric constant, L, describing the equilibrium between the 2 unligated forms is different for each ligand. This implies that at least 3 allosteric forms are needed to characterize the binding of oxygen and carbon monoxide to this hemocyanin.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate.

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO₂ for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ($\text{Δlog}\text{P}{}_{30}\text{ΔpH}$) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in $\text{Δlog}\text{P}{}_{50}\text{ΔpH}\text{~ 0.3}$). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.     

Carp hemoglobin: Functional analysis and thermodynamics of precise oxygen equilibria according to the simple sequential model of Koshland,Némethy and Filmer

Precise oxygen equilibrium curves for carp hemoglobin were determined at 15 °C in bis-Tris buffer, and in phosphate buffer in the presence and absence of P₆-inositol, and at various temperatures in phosphate buffer. Parameters of the Koshland, Némethy and Filmer (1966) (KNF) simple, sequential models (square and tetrahedral) were estimated by non-linear least-square fit of the experimental data to Hill plots. Non-, negative and positive co-operativity can be fitted by the KNF models. Considering equilibrium arguments, $\text{K}{}^2{}_{\mathrm{<mtext>AB</mtext>}}\text{K}{}_{\mathrm{<mtext>BB</mtext>}}$, the KNF parameter governing the co-operativity of the system, predicts a symmetry conserved mode of action in regions of high, positive co-operativity, and a symmetry non-conserved mode of action in regions of low, non- or negative co-operativity. The simple, sequential, square KNF model fits better the Hill plots than does the simple, sequential, tetrahedral KNF model. From the effect of temperature on carp hemoglobin in phosphate buffer, the heats and entropies of the subunit interaction parameter, $\text{K}{}^2{}_{\mathrm{<mtext>AB</mtext>}}\text{K}{}_{\mathrm{<mtext>BB</mtext>}}$, and of the oxygenation parameters, K_BBK_xBK_tAB and $\text{K}{}^{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}{}_{\mathrm{<mtext>BB</mtext>}}\text{K}{}_{\mathrm{<mtext>xB</mtext>}}\text{K}{}_{\mathrm{<mtext>tAB</mtext>}}$, for the square and tetrahedral models, respectively, were calculated and show the square model to account well for previously published data on the carp hemoglobin molecule. This study indicates that the KNF model, in its simplest form, is capable of explaining many of the functional properties of cooperative systems, as opposed to the Monod, Wyman and Changeux (1965) model which seems only to be a special case of the KNF model in regions of high, positive co-operativity.     

Hemoglobin Dallas (α97(G4)Asn→Lys):functional characterization of a high oxygen affinity natural mutant

Hemoglobin Dallas, an α-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{mmHg at pH}\text{7.3}\text{and}\text{20°C), diminished cooperativity (0n,}\text{the Hill coefficient}\text{= 1.7}$) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20°C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be atttributed to a more ‘relaxed’ T-state.     

Stimulation of minor fetal hemoglobin synthesis in cord blood reticulocytes by butyrate

As it had been shown that sodium butyrate promoted hyperacetylation of nuclear histones the effect of 5, 10, and 50 mM sodium butyrate on the $\text{in}\text{vitro}$ synthesis of Hb F_Ic (an acetylated component of Hb F) and Hb F in cord blood reticulocytes was studied. The presence of 5 mM butyrate showed a significant increase in the production of Hb F_Ic, whereas Hb F synthesis was unaffected. Higher concentrations of butyrate, however, had no stimulatory effect on Hb F_Ic synthesis. Results of pulse-chase studies indicated that sodium butyrate inhibited the turnover of preformed Hb F_Ic, probably by inhibiting deacetylase.     

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.     

Correlation of mitochondrial cytochrome concentration and activity to oxygen availability in the newborn.

Effect of $\text{in vivo}$ oxygenation on mitochondrial respiratory chain capacity was studied in newborn puppies. Heart mitochondria were isolated from 0 to 7 days. As the arterial PO₂ rises sharply during the first 3–4 days, State 3 respiratory capacity and Ca⁺⁺ transport activity decrease linearly. Cytochrome $\text{c}$ and $\text{a + a}$₃ concentrations increase rapidly. Thus the enzymatic turnover of the respiratory chain decreases to one-fifth of the fetal term value in 4 days. These data are in agreement with earlier results indicating a strong controlling effect of $\text{in vivo}$ oxygenation on mitochondrial respiratory capacity.