Valency Hybrids Research Articles

Heterogeneity between Two α Subunits of α2β2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the β subunit of α2β2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)β(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2β2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)β(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)β(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)β(Fe3+)β(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2β2 tetramer changes the other α subunit to the R structure.

The alpha 1 beta 1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen.

When the alpha and beta chains were separated from human oxyhemoglobin (HbO(2)), each individual chain was oxidized easily to the ferric form, their rates being almost the same with a very strong acid-catalysis. In the HbO(2) tetramer, on the other hand, both chains become considerably resistant to autoxidation over a wide range of pH values (pH 5-11). Moreover, HbA showed a biphasic autoxidation curve containing the two rate constants, i.e. k(f) for the fast oxidation due to the alpha chains, and k(s) for the slow oxidation to the beta chains. The k(f)/k(s) ratio increased from 3.2 at pH 7.5-7.3 at pH 5.8, but became 1 : 1 at pH values higher than 8.5. In the present work, we used the valency hybrid tetramers such as (alpha(3+))2(beta O(2))(2) and (alpha O(2)(2)(beta(3+))(2), and demonstrated that the autoxidation rate of either the alpha or beta chains (when O2- ligated) is independent of the valency state of the corresponding counterpart chains. From these results, we have concluded that the formation of the alpha 1 beta 1 or alpha 2 beta 2 contact suppresses remarkably the autoxidation rate of the beta chain and thus plays a key role in stabilizing the HbO(2) tetramer. Its mechanistic details were also given in terms of a nucleophilic displacement of O(2)(-) from the FeO(2) center, and the emphasis was placed on the proton-catalyzed process performed by the distal histidine residue.

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.

Reactions between nitric oxide and haemoglobin under physiological conditions

The tenet of high-affinity nitric oxide (NO) binding to a haemoglobin (Hb) has shaped our view of haem proteins and of small diffusible signaling molecules. Specifically, NO binds rapidly to haem iron in Hb (k approximately 10[7] M[-1] s[-1]) and once bound, the NO activity is largely irretrievable (Kd approximately 10[-5] s[-1]); the binding is purportedly so tight as to be unaffected by O2 or CO. However, these general principles do not consider the allosteric state of Hb or the nature of the allosteric effector, and they mostly derive from the functional behaviour of fully nitrosylated Hb, whereas Hb is only partially nitrosylated in vivo. Here we show that oxygen drives the conversion of nitrosylhaemoglobin in the 'tense' T (or partially nitrosylated, deoxy) structure to S-nitrosohaemoglobin in the 'relaxed' R (or ligand-bound, oxy) structure. In the absence of oxygen, nitroxyl anion (NO-) is liberated in a reaction producing methaemoglobin. The yields of both S-nitrosohaemoglobin and methaemoglobin are dependent on the NO/Hb ratio. These newly discovered reactions elucidate mechanisms underlying NO function in the respiratory cycle, and provide insight into the aetiology of S-nitrosothiols, methaemoglobin and its related valency hybrids. Mechanistic reexamination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

In vivoDetection of Nitric Oxide and NOxSpecies Usingex vivoElectron Paramagnetic Resonance Spectroscopy

Nitric oxide is a paramagnetic molecule that exhibits high affinity for heme proteins, forming a mixture of nitrosylated valency hybrids that can be precisely characterized using low-temperature electron paramagnetic resonance spectroscopy. In red blood cells, nitric oxide will form paramagnetic complexes with deoxyhemoglobin or be oxidized to nitrite and nitrate by oxyhemoglobin with formation of paramagnetic methemoglobin. We have developed a technique to monitor temporal changes in local nitric oxide productionin vivo,by sampling arterial and venous blood across a tissue bed and measuring changes in hemoglobin–nitric oxide and methemoglobin concentrationex vivousing electron paramagnetic resonance spectroscopy. Changes in local nitric oxide concentration are further clarified by quantifying plasma nitrite/nitrate levels. With these techniques, we have observed that whole body hyperthermia increases portal venous concentrations of hemoglobin–nitric oxide and organic radicals that are consistent with enhanced nitric oxide synthase activity, cytotoxic leukocyte activity, and cellular oxidative stress in splanchnic tissues.

Reexamination of the hyper thermodynamic stability of asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): no preferentially populating asymmetric hybrid at equilibrium.

It has been reported that hybridization of the equimolar mixture of cyanomethemoglobin and deoxyhemoglobin through dimer exchange reaction results in establishment of an approximately binomial (1:2:1) equilibrium distribution of these parental hemoglobins and their hybrid molecule, (alpha+CN-beta+CN-)(alpha beta), within several days under anaerobic conditions at pH 7.4, 21.5 degrees C, leading to a hyper (i.e., about 170 times) thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) relative to the stability of the other diliganded species at pH 7.4, 21.5 degrees C [Daugherty, M. A., Shea, M. A., Johnson, J. A., LiCata, V. J., Turner, G. J., & Ackers, G. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. To examine whether the published "binomiality" for this deoxy-cyanomet hybrid system really reflects the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta), we used a binomial (1:2:1) equilibrium distribution of the equimolar mixture of cyanomethemoglobin and fully oxygenated hemoglobin as a starting condition, and then this system was rapidly deoxygenated. We found that the relative population of the hybrid was reduced to 8.6% of the total upon deoxygenation. It was also found that the hybridization experiment under anaerobic conditions was not allowed to continue for a long time due to a valency exchange reaction between deoxy and cyanomet derivatives. For instance, a 48 h incubation resulted in the oxidation of 44% of Fe2+ to Fe3+ hemes in the original deoxyhemoglobin and the reduction of 42% of Fe3+ to Fe2+ hemes in the original cyanomethemoglobin. These results suggest that a real distribution of the deoxy-cyanomet hybrid system at equilibrium is fairly far from 1:2:1 (binomial distribution), and the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) is less than one-tenth of the hyperstability previously reported. In addition, most of the previous results on deoxy-cyanomet valency hybrids placed under long anaerobic conditions should be subject to reexamination due to possible valency exchange reactions.

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.

Asymmetric Hemoglobin Hybrids

We have investigated the functional properties of hemoglobin (Hb) valency hybrids, and specifically whether it makes a difference if the oxidized subunits are on the same dimer (asymmetric hybrid) or not. CO recombination kinetics were used to probe the allosteric equilibrium of tetramers with two oxidized subunits. Asymmetric hybrids were prepared by mixing HbCO with a large excess of cyano-metHb; dimer exchange occurs in the order of seconds, producing a population of fully liganded [dimer-CO/dimer-CN] hybrids; these hybrids could then be photolysed to study CO rebinding to the doubly liganded [dimer-deoxy/dimer-CN] species. Before mixing, the HbCO samples typically show 30 to 50% slow phase, characteristic of deoxy (or T-state) Hb. Addition of HbCN to these samples decreased the slow fraction. The higher the ratio of HbCN to HbCO, the less slow phase was observed, with about 5% slow phase at a ratio of 10:1. This would indicate that the photoproduct [deoxy-dimer/dimer-CN] is not predominantly in the low-affinity (T-state) conformation. We did not observe the difference between asymmetric and symmetric hybrids expected from published studies. The difference between the present flash photolysis results and the published equilibrium studies could be due to a kinetic factor: if the conversion to the T-state is slow after photolysis, then the biomolecular kinetics will reflect the pre-flash conditions of fully liganded (R-state) hemoglobin.

Analysis of Hemoglobin Derivatives by Capillary Isoelectric Focusing and Its Application in the Antidotal Research of Cyanide Poisoning

Cyanide toxicity can be reduced by the use of methemoglobin (MetHb) formers, and antidotal dosage is based on the extent of MetHb formation. Hemoglobin and ferrihemoglobin (MetHb, hemimethemoglobins α3+β2+and α2+β3+, tetracyanmethemoglobin, and dicyanmethemoglobin) concentrations in human, pig, and mouse blood were determined after separation by isoelectric focusing with an octyl-bonded capillary. The predominant species formed in blood when MetHb formers, such as potassium ferricyanide, hydroxylamine, sodium nitrite, and 4-dimethylaminophenol (DMAP), added at molar ratios ranging from 1:10 to 1:1 to hemoglobin, are the valency hybrid intermediates α3+β2+and α2+β3+. In the detoxication of cyanide with methemoglobin, an intermediate dicyanhemimethemoglobin was demonstrated to be the predominant species in the formation of tetracyanmethemoglobin. Complex mixtures of hemoglobin derivatives were observed with DMAP at 1:1 or greater molar ratio to hemoglobin. Comparison of the MetHb values obtained with a hemoxometer indicated that the valency hybrids were measured as MetHb and the values of oxidized hemoglobin were overestimated. In cyanide poisoning, incorrect dosages of MetHb formers could be calculated, and misinterpretation of MetHb data would result from methods that fail to discriminate among the various species of MetHb.

Oxygen Equilibrium Properties of Chromium(III)-Iron(II) Hybrid Hemoglobins

Cr(III)-Fe(II) hybrid hemoglobins, alpha 2(Cr) beta 2(Fe) and alpha 2(Fe) beta 2(Cr), in which hemes in either the alpha- or beta-subunits were substituted with chromium(III) protoporphyrin IX (Cr(III)(PPIX), were prepared and characterized by oxygen equilibrium measurements. Because Cr(III)PPIX binds neither oxygen molecules nor carbon monoxide, the oxygen equilibrium properties of Fe(II) subunits within these hybrids can be analyzed by a two-step oxygen equilibrium scheme. The oxygen equilibrium constants for both hybrids at the second oxygenation step agree with those for human adult hemoglobin at the last oxygenation step (at pH 6.5-8.4 with an without inositol hexaphosphate at 25 degrees C). The similarity between the effects of the Cr(III)PPIX and each subunits' oxygeme on the oxygen equilibrium properties of the counterpart Fe(II) subunits within hemoglobin indicate the utility of Cr(III)PPIX as a model for a permanently oxygenated heme within the hemoglobin molecule. We found that Cr(III)-Fe(II) hybrid hemoglobins have several advantages over cyanomet valency hybrid hemoglobins, which have been frequently used as a model system for partially oxygenated hemoglobins. In contrast to cyanomet heme, Cr(III)PPIX within hemoglobin is not subject to reduction with dithionite or enzymatic reduction systems. Therefore, we could obtain more accurate and reasonable oxygen equilibrium curves of Cr(III)-Fe(II) hybrids in the presence of an enzymatic reduction system, and we could obtain single crystals of deoxy-alpha 2(Cr) beta 2(Fe) when grown in low salt solution in the presence of polyethylene glycol 1000 and 50 mM dithionite.

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.

Isolation and Stability of Partially Oxidized Intermediates of Carp Hemoglobin: Kinetics of CO Binding to the Mono- and Triferric Species

The monoliganded and triliganded forms of the asymmetric valency hybrids of carp hemoglobin were isolated using high-performance liquid chromatography. These partially oxidized hybrids were shown to be sufficiently stable to permit the measurement of the kinetics of CO binding. The effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO binding to the fully ferrous molecule. To a first approximation, the kinetic behavior of the monoferric derivative was consistent with a small shift in the T<==>R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conformational equilibrium in favor of the R state. The kinetic behavior of the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presence of cyanide on the ferric hemes than by water. Not all of the data could be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivative at low pH in the presence of inositol hexaphosphate.

17] Preparation of intramolecularly cross-linked hemoglobins

Publisher Summary This chapter discusses the preparation of intramolecularly cross-linked hemoglobins. Hemoglobin (Hb), although normally a tetramer, readily undergoes dissociation to form αβ dimers. By cross-linking the molecule intramolecularly, this process can be blocked. This has become important both for basic structure-function studies of Hb and for the development of Hb derivatives useful as blood substitutes. Preparation of mixed metal and valency hybrids has made it possible to isolate stable species representing intermediate ligation states of Hb; these have yielded important new insights into the linkage between ligand binding and the structural and energetic changes that underlie cooperativity. Cross-linking is also important from the standpoint of developing Hb derivatives to serve as blood substitutes. The αβ dimers having a molecular weight of only 32,000 are readily filtered from the circulation by the kidneys. As a result, unmodified Hb has a very short plasma half-life, and the massive hemoglobinuria that results poses the risk of renal injury. It is essential to cross-link the molecule intramolecularly to prevent this from occurring.

6] Allosteric equilibrium measurements with hemoglobin valency hybrids

Publisher Summary This chapter discusses the allosteric equilibrium measurements with hemoglobin (Hb) valency hybrids samples.There is a need to correct for the influence of a slightly oxidized sample. Although the oxidation levels can usually be maintained below 5%, this level is worse than it seems when dealing with a tetrameric system. This problem may be aggravated in Hb mutants, natural or artificial, in which the rate of autoxidation is increased. Thus, an understanding of how ferric subunits affect their neighbors within an Hb tetramer is important in order to compare the ligand-binding parameters of different samples. The use of highly oxidized solutions provides another method to study the triply liganded form. With different ferric ligands and the use of external effectors– whether the two-state model is still operational at the triply liganded level can be determined. By comparing different ferric ligands with varying amounts of high- and low-spin ferric hemes, the coupling of the spin state and the allosteric equilibrium can be studied.

20] Double mixing methods for kinetic studies of ligand binding in partially liganded intermediates of hemoglobin

This chapter focuses on the differences between the two methods of double mixing stopped-flow and single mixing, the type of reactions that can be studied by the double mixing method, experimental design, data treatment, and a brief discussion of the results obtained. Unless otherwise mentioned, all examples discussed in the chapter are for normal human hemoglobin (Hb). The double-mixing method , along with the micro peroxidase method for studying carbon mono oxide (CO) dissociation reactions and the high pressure liquid chromatography (HPLC) method for preparation of the valency hybrids, allows for the study of kinetics of CO association with, and dissociation from, partially liganded intermediates of Hb. All of these studies so far have been made by only two groups and, therefore, a certain degree of bias in perspective may have been unavoidable, particularly in formulating reaction schemes to fit the data. These studies have been made using a first-generation double mixing stopped-flow spectrophotometer with limited capabilities, such as a long dead time, use of large sample volumes for each kinetic shot, and some probability of leading-edge contamination at longer aging times. More information and precision can be obtained by using state-of-the-art double-mixing instruments.

Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.

Model calculations of chemical interactions. Part 4.—Directed valency and analysis of intramolecular interactions with redundant basis sets

Directed valency hybrids are obtained starting with a double-zeta-plus polarization basis set. The original atomic basis set (6-31G*/5d in our example) is split into a set of high-occupancy hybrids, a set of low-occupancy hybrids and a set of ‘pure’ polarization functions which, unlike hybrids, are not ‘assigned’ to specific molecular fragments (bonds, lone pairs, etc.) but to the molecule as a whole. Analysis of intramolecular interactions according to a procedure originally formulated for minimal basis sets reveals that these hybrids retain the essential features and meanings of those obtained in minimal basis sets, although they introduce the effects of electron delocalization from high- to low-occupancy hybrids of different fragments.

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.

Chemicals Reacting with Various Forms of Hemoglobin: Biological Significance, Mechanisms, and Determination

The clinical chemistry of various forms of hemoglobin occupies a large fraction of the total activities of hospital and forensic science laboratories. Some of the potential pitfalls are reviewed here along with an account of the accidental discovery of two novel chemical forms. Human or mouse red blood cells were exposed to excess sodium nitrite to convert the intracellular pigment to methemoglobin. When these were subsequently incubated in Krebs-Ringer-phosphate-glucose medium, pH 7.4 at 37 degrees C under nitrogen and in the presence of various concentrations of methylene blue, a blood pigment was generated in high yield which had unique properties. In lysates, the pigment was stable in air, and it could be maintained in liquid nitrogen for as long as a year without deterioration. The pigment had properties different from those of oxyhemoglobin, deoxyhemoglobin, methemoglobin, or carboxyhemoglobin. After separation by isoelectric focusing, the pigment gave a strong signal on electron paramagnetic resonance (EPR) spectroscopy. The other forms of hemoglobin given above are EPR-silent. The pigment was eventually identified as the nitrosylated valency hybrid species, (alpha 2+ beta 3+)2(NO)2. The corresponding species, (alpha 3+ beta 2+)2(NO)2, has similar properties. These species apparently owe their unusual stability in air to the presence of the oxidized subunits in the same tetramer.