[SaO2 and PaO2 mismatch: Do not forget hemoglobinopathy]. / Discordance entre SaO2 ­ PaO2 : ne pas oublier les hémoglobinopathies.

INTRODUCTION: Different clinico-biological parameters are used to estimate the amount of oxygen available for the organism. Oxygen saturation measured with pulse oxymetry (SpO2), oxygen saturation of arterial blood (SaO2) and oxygen partial pressure of the arterial blood (PaO2) are the most commonly used. CASE REPORT: We report the case of a patient admitted for investigation of respiratory failure in the context of chronic dyspnea of effort. SpO2 and SaO2 were decreased, though the PaO2 was normal. This mismatch between oxygen saturation and PaO2 led to the diagnosis of hemoglobinopathy (Bassett hemoglobin). CONCLUSION: The diagnosis of hemoglobinopathy should be considered in cases of oxygen desaturation with normal respiratory and cardiac investigations. There are no reasons to prescribe long-term oxygen to these patients.

Heme binds to factor VIII and inhibits its interaction with activated factor IX.

BACKGROUND: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme-binding proteins. Free heme has been proposed to affect coagulation and has been co-purified with the factor VIII (FVIII)-von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. OBJECTIVES: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. METHODS AND RESULTS: Heme bound to several sites on FVIII with high apparent affinity. Heme-binding inhibited FVIII procoagulant activity in a dose-dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme-mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme-binding protein known for its protective activity in hemolytic conditions. CONCLUSIONS: Our data identify FVIII as a novel heme-binding protein. Occupation of high affinity heme-binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme-mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.

Short insertion in a hemoglobin chain: Hb Esch, an unstable alpha1 variant with duplication of the sequence Ala65-Leu-Thr-Asn68.

Hemoglobin (Hb) Esch, is an alpha1 variant, expressed at less than 5%, resulting from the duplication of the 12 nucleotides corresponding to CD65 through 68. The effect of this insertion is the repetition of the sequence Ala-Leu-Thr-Asn, which corresponds to the last turn of helix E. In this variant the presence of a one-turn elongated helix E causes instability and increased ligand affinity. Hb Esch was characterized by DNA sequencing and confirmed by electrospray mass spectrometry. Functional studies were performed by flash photolysis measurements on a fraction isolated by flatbed isoelectric focusing, which was enriched in the abnormal hemoglobin. Similar to other alpha chain variants due to short insertion (or deletion), Hb Esch probably results from a slipped mispairing mechanism. The stability of such modified proteins depends upon the region which is added or deleted and usually is more stable when involving a flexible loop or complete helix turn(s) near by.

Electron transfer kinetics between hemoglobin subunits.

The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, alpha or beta, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25 degrees C, the Hb solution (0.1 mm heme) approaches a form with about 60% of beta chains reduced, indicating a faster transfer rate in the direction alpha to beta. There was no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized cross-linked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte.

Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family.

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O(2) supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe(2+)) form, indicating a His-Fe(2+)-His binding scheme. O(2) or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O(2) and CO binding is suggested to be slow in vivo. Because of this competition, the observed O(2) affinity of recombinant human neuroglobin is average (1 torr at 37 degrees C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 degrees C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E'(o) = -129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.

Heme photolysis occurs by ultrafast excited state metal-to-ring charge transfer.

Ultrafast time-resolved resonance Raman spectra of carbonmonoxy hemoglobin (Hb), nitroxy Hb, and deoxy Hb are compared to determine excited state decay mechanisms for both ligated and unligated hemes. Transient absorption and Raman data provide evidence for a sequential photophysical relaxation pathway common to both ligated and unligated forms of Hb* (photolyzed heme), in which the excited state 1Q decays sequentially: 1Q-->Hb*I-->Hb*II-->Hb ground state. Consistent with the observed kinetics, the lifetimes of these states are <50 fs, approximately 300 fs, and approximately 3 ps for 1Q, Hb*I, and Hb*II, respectively. The transient absorption data support the hypothesis that the Hb*I state results from an ultrafast iron-to-porphyrin ring charge transfer process. The Hb*II state arises from porphyrin ring-to-iron back charge transfer to produce a porphyrin ground state configuration a nonequilibrium iron d-orbital population. Equatorial d-pi* back-bonding of the heme iron to the porphyrin during the lifetime of the Hb*II state accounts for the time-resolved resonance Raman shifts on the approximately 3 ps time scale. The proposed photophysical pathway suggests that iron-to-ring charge transfer is the key event in the mechanism of photolysis of diatomic ligands following a porphyrin ring pi-pi* transition.

Modulation of the oxygen affinity of cobalt-porphyrin by globin.

We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.

Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T.

In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb. We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T. In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations. In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations. However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation. Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM. Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate. These mutations are thus of interest in developing a Hb-based blood substitute.

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.

Structural, functional, and genetic characterization of Gastrophilus hemoglobin.

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.

CO binding and valency exchange in asymmetric Hb hybrids.

There remains a major controversy concerning the properties of asymmetric hemoglobin hybrids, that is, doubly liganded tetramers consisting of an unliganded dimer and a liganded dimer. Different experimental evidence leads to opposing conclusions. Based on dimer-tetramer equilibrium studies, special "T-like" properties were assigned to this hybrid (species 21), while the other biliganded tetramers were considered as similar to fully liganded Hb [Ackers et al. (1992) Science 255, 54-63]. We report here results for three types of experiment. In the first, the asymmetric hybrids are produced by photodissociating CO ligands from [dimer-CO/dimer-azido-met] hybrids. Since the CO association rates differ by over an order of magnitude for the two allosteric states, the CO kinetics are a sensitive probe of the tetramer conformation. The results show mainly rapid R-like kinetics for CO rebinding to the asymmetric hybrids. The second technique employs a stopped-flow apparatus to obtain a higher percentage and a longer equilibration time of the asymmetric hybrid. In this case, sodium dithionite is used to remove oxygen from a solution containing [dimer-oxy/dimer-azido-met] hybrids. After a fixed delay (but before loss of azide ligands), a second mixing with a buffer equilibrated under CO allows observation of CO binding to species 21. As for the flash measurements, the kinetics show predominantly rapid CO binding, typical of the liganded (R-state) tetramer. The rapid CO binding is not in agreement with the predictions of a T-like conformation for species 21. One possible explanation is that the long incubation times used to study the dimer-tetramer equilibrium do not lead to a stable asymmetric hybrid, but rather a random distribution of oxidized subunits due to electron transfer between the iron atoms of the subunits [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We have repeated these experiments and confirm the valency exchange in a mixture of Hb A and S (or C) parent forms, as evidenced by compensating amounts of oxidation or reduction of the Hb parents.

Intracranial hypotension presenting with severe encephalopathy. Case report.

A patient with severe and protracted symptoms from intracranial hypotension is described. The patient's presentation was marked by diffuse encephalopathy and profound depression of consciousness. This case report expands the presently known clinical spectrum of this uncommon and generally benign illness. The clinical and laboratory findings typically observed in the syndrome of intracranial hypotension are outlined. The pathophysiological mechanisms of the phenomenon are briefly discussed. Intracranial hypotension is a potentially severe illness with specific treatments that are distinct from the treatment of most neurological diseases. Three cardinal features--postural headache, pachymeningitis, and descent of midline cerebral structures--should prompt the diagnosis.

Trematode hemoglobins show exceptionally high oxygen affinity.

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Thermodynamic studies on the equilibrium properties of a series of recombinant betaW37 hemoglobin mutants.

In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].

Fluorescent effector as a probe of the allosteric equilibrium in methemoglobin.

A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetrisulfonate (HPT), was used as a probe of the allosteric equilibrium of methemoglobin. Like DPG, HPT binds, one per tetramer, with a higher affinity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is quenched by energy transfer to the hemes. HPT can thus serve as a probe of the conformational state of the hemoglobin tetramer: a higher quenching indicates a stronger binding and therefore, more of the deoxy conformation. Since HPT binds to the same site as DPG, it can be displaced by DPG in order to determine the fluorescence intensity of the free HPT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluorescence (more of the deoxy state) than low spin cyano-metHb. The aquo-metHb samples showed a reversion to the oxyHb conformation above pH 7, as expected due to the acid-alkaline transition forming hydroxy-metHb. Effectors such as bezafibrate, which do not bind to the same site as DPG, show an increase in the deoxy-like characteristics.

Identifying the conformational state of bi-liganded haemoglobin.

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.

False-positive behavior with the dP/dt sensing pacemaker: a rare complication of a physiological sensor.

As with "nonphysiological" devices, sensors that directly measure physiological variables have the potential to measure unexpected signals and for the physiological parameter being measured to respond in an unexpected manner. We present the case of a dP/dt sensing pacing system that functioned normally for 2 months and then developed upper rate behavior due to the sensing of a high frequency artifact on the pressure recording. Our case and others cited reinforce the need for future physiological rate responsive pacemakers to incorporate a second sensor to provide for backup rate response in cases of inappropriate rate response.

Recombinant [Phe(beta)63]hemoglobin shows rapid oxidation of the beta chains and low-affinity, non-cooperative oxygen binding to the alpha subunits.

We have engineered alpha2beta2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the beta chains to a phenylalanine. The mutant tetramer binds four high-affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it binds only two low-affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated beta subunits, is over 1000-fold faster than for Hb A. The mutant Hb exhibits non-cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (alpha met beta)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280-282]. Flash-photolysis experiments also indicate non-cooperative behaviour: the CO-recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (alpha(CO)metbeta)2 valency hybrid or the (alphaCO betaCO)2 form, suggesting mainly geminate CO-recombination kinetics to the beta chains. EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six-coordinated iron with low-spin character. The EPR and resonance Raman spectra for the mutated beta subunits demonstrate the presence of a high-spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated beta subunits are penta-coordinated. The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.