Control of the allosteric equilibrium of hemoglobin by cross-linking agents

Abstract

The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with α Val 1 or involve a cross-link between α Val 1 and α Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 μM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange.

Many novel types of modified hemoglobin (Hb) have been prepared based on different strategies, including site-directed mutagenesis (Baudin et al. 1992; Looker et al. 1992; Bunn 1993), addition of external effectors (Lalezari et al. 1990), polymerization, and cross-linking (Snyder et al. 1987). In some cases the external constraints can maintain the Hb in the deoxy conformation even after all four ligands are bound (Mozzarelli et al. 1991; Shibayama and Saigo 1995). There is an increasing interest in understanding and modifying the ligand binding properties of hemoglobin, in view of preparing artificial hemoglobins that can be used as blood substitutes (Winslow 1992; Jones 1995). The use of Hb solutions, as a substitute for red blood cells, requires the modification of several parameters. Much progress has been made in regulating the Hb affinity for oxygen, either by introduction of mutations or use of cross-linking agents. The size of the transporter can be modified by cross-linking the dimers to form stable tetramers or by formation of larger units through tetramer polymerization.

Less progress has been made in the control of the rate of autoxidation, and unfortunately the oxygen affinity, tetramer stability, and the oxidation cannot be regulated independently. Hemoglobins with lower oxygen affinity tend to autoxidize more rapidly (Brantley et al. 1993), and dissociation of Hb tetramers yields high oxygen affinity dimers with a greatly increased rate of oxidation (Zhang et al. 1991; Griffon et al. 1998). Oxidized subunits do not bind oxygen and due to the allosteric nature of ligand binding they will influence the properties of the remaining ferrous subunits. Taking into account both effects, the loss in oxygen transport is nearly twice the loss of oxygen binding sites; that is, oxidation of 10% of the hemes results in nearly a 20% loss in oxygen delivery (Marden et al. 1995). Thus the oxidation rate will limit the useful lifetime of a Hb-based blood substitute.

A large range of effects on the oxygen-binding properties of Hb was shown in the previous studies of a series (Fig. 1 ▶) of bis-aldehyde bis-acid allosteric effectors that cross-link Hb via Val 1 of the α₁ subunit with Lys 99 of α₂ (Fig. 2 ▶) in the opposing dimer (Abraham et al. 1995) and mono-aldehyde allosteric effectors that react at α Val 1 but lack a second aldehyde group to cross-link the dimers (Boyiri et al. 1995). In this study of ligand-binding kinetics, the allosteric equilibrium of these modified hemoglobins has been further evaluated.

Fig. 1.

Chemical structure of hemoglobin (Hb) bis-aldehyde cross-linking agents (series 1) and mono-aldehyde modulators (series 3 and other compounds). The overall length can be varied to generate a family of cross-linked Hbs; in this study n = 1–4 for Hb 1a–1d.

Fig. 2.

Crystallographic image of cross-linking site of Hb 1c, where n = 3 for series 1 in Fig. 1 ▶. A double-bridge is formed by two molecules of 1c (labeled "TB36" at the center of the image), which link the amino-terminal amino nitrogen of one α subunit with the ɛ-amino nitrogen of Lys 99 of the opposite α subunit.

By variation of the length of the cross-linker, by the number of CH₂ groups (Fig. 1 ▶), a variation of over an order of magnitude in the oxygen affinity was obtained (Abraham et al. 1995; Boyiri et al. 1995). In some cases, the cross-linked Hb showed a low (T-like) oxygen affinity with little cooperativity, indicating a late switchover point, with the tetramer being mainly in the low affinity conformation, even with three ligands bound. This result leads to the question as to whether there may be a certain fraction of T-state conformation for the fully liganded form, which is difficult to determine from the oxygen equilibrium curves alone, due to the large compensation between the fitting parameters L and K_R whenever the switchover point is >3 ligands (Marden et al. 1990). In such cases, the binding of the fourth ligand is no longer between two pure R-states, and the observed affinity for the last ligand is "contaminated" by a certain fraction of T-state.

The kinetic studies can be used to study the properties of the fully liganded form. In analogy to equilibrium studies that show two ligand affinities, the bimolecular kinetics display two association rates, with ligand binding to the deoxy conformations corresponding to the slower phase. Interpretation of equilibrium and kinetic data are generally well correlated in their dependence on the percent dissociation, pH, allosteric effectors, and mutations of the protein sequence. Both methods can be used to probe the allosteric equilibrium of the partially liganded forms. One advantage of the flash photolysis technique is that at low photodissociation levels the main photoproduct is triply liganded Hb and one can then isolate the reaction for the binding of the fourth ligand.

The timescale of the ligand rebinding is also relevant for measurements of the properties of the two conformations. The geminate phase, which occurs on the nanosecond timescale in aqueous solutions, is generally complete before the R to T transition and therefore has rates determined by the preflash conformation. For fully liganded HbA, the protein is typically in the R-state and one observes about 50% geminate phase at 25°C. However, for conditions that favor the deoxy conformation, the rate and relative amplitude of geminate phase are much lower (Sawicki and Gibson 1979; Marden et al. 1987; Murray et al. 1988). A shift towards the T-state conformation results in slower ligand-rebinding rates and a larger fraction of photolyzed ligands that escape to the solvent and subsequently rebind via the bimolecular phase. Thus the fraction of the kinetics occurring at the bimolecular phase can also be a probe of the allosteric conformation.

Results

The kinetics of CO recombination to the modified hemoglobins are shown in Figure 3 ▶. The curves are typical of hemoglobin, displaying two bimolecular phases characteristic of the two allosteric conformations. Although the rates are similar for the various cross-linked forms, the main difference is in the fraction rebinding with the slow rate (Table 1), indicating a shift in the allosteric equilibrium.

Fig. 3.

Bimolecular kinetics of CO recombination to modified hemoglobins at pH 7, 25°C. The kinetics are biphasic, with the slow-phase characteristic of CO binding to the deoxy (T-state) conformation. Cross-linkers 1a, 1b, and 1c (n = 1, 2, and 3, respectively, for the number of CH₂ groups; see Fig. 1 ▶, series 1) are di-aldehydes that bind to both dimers. The modifiers 3d and 5FSA are mono-aldehydes that form only one covalent bond with the protein. The rates for the rapid and slow phases do not change by more than a factor of 2 relative to HbA; the dominant shift is in the allosteric equilibrium, which changes the fraction of slow phase.

Table 1.

Ligand binding to cross-linked hemoglobin

	O2 equilibrium		Oxidation	CO photolysis kinetics % slow phase
	n50	P50 (mm Hg)	K/KHbA	φ	f = 0.5	f = 0.05
HbA (control)	2.8	4.5	1.0	0.50	18	<1
bis-aldehyde cross-links
1a n = 1	1.5	24	1.3	0.43	66	14
1b n = 2	1.4	40	1.6	0.70	71	45
1c n = 3	1.5	27	1.3	0.60	60	57
1d n = 4	1.6	33	0.9	0.67	67	20
Mono-aldehyde modulators
3d n = 4	2.0	10	0.8	0.54	30	5
4-CBA	2.3	13	1.1	0.44	26	<1
2-BF	2.4	9	1.1	0.49	24	<1
5-FSA	2.5	8	1.4	0.47	23	<1
2,4CBF	2.2	5	0.5	0.53	18	<1
DMHB	1.8	2	0.6	0.44	19	<1

Only the bis-aldehyde molecules (series 1; see Fig. 1 ▶) provide a covalent bond to both dimers; n is the number of CH₂ groups between the aromatic rings. Oxygen equilibrium parameters at pH 7.2, 25°C, 100 mM NaCl (Abraham et al. 1995; Boyiri et al. 1995); oxidation rates were measured at pH 7, 37°C. CO binding kinetics were determined at pH 7, 25°C; φ is the photoyield for the bimolecular phase (4 μsec after photodissociation). Binding of the fourth ligand is best observed at a low-Fraction photodissociation, f = 0.05, in which the main photoproduct is triply liganded hemoglobin (Hb).

Kinetics vs. fraction dissociation

Variation of the laser energy is a useful experimental parameter for probing the allosteric equilibrium. At high photolysis levels, producing mainly deoxy or singly liganded tetramers, a large fraction of the tetramers will convert to the T-state. At sufficiently low laser energies, the main photoproduct is the triply liganded form, which, for unmodified HbA, remains in the R-state. For native Hb, a large variation in the slow fraction is observed upon variation of the fraction dissociated. Several cross-linked Hbs studied here showed little change (Fig. 4 ▶). In extreme cases such as Hb 1b, covalently cross-linked between the α chains by a bis-aldehyde (see Fig. 1 ▶, series 1, with n = 2), the kinetics showed about 80% slow phase at high photolysis levels, and even at low dissociation levels the slow kinetics accounted for nearly half of the bimolecular signal (Table 1). For Hb 1c (see Fig. 1 ▶, series 1, with n = 3, and Fig. 2 ▶), with the same cross-linking agent as for Hb 1b except for a longer chain, the fraction of slow phase was practically independent of the laser energy.

Fig. 4.

CO recombination to hemoglobin A (HbA) and cross-linked Hb 1c at high and low photodissociation levels, at pH 7, 25°C. At low-dissociation levels, in which the recombination is mainly to the triply liganded form, HbA shows a decrease in the fraction of slow (T-state) phase, whereas Hb 1c shows little change as a function of laser energy.

The rates for the rapidly binding conformation (R-state) are best determined at higher CO concentrations and lower photodissociation level; the CO on-rates did not deviate by more than 20% relative to HbA. For the slower phase, a decrease in rate as much as a factor of 2 was observed.

Switchover point

For cases showing mainly T-like CO recombination kinetics, one can ask whether Hb is in the T-state even with four ligands. One must dissociate at least one ligand to observe the kinetics; if the kinetics are T-like even at low dissociation levels, one can conclude that the tetramer adopts the T-state with three ligands bound but not necessarily with all four ligands.

In the case of native HbA, the Hb(CO)₄ tetramer is in the R-state structure before photolysis, and conversion to the deoxy (T-state) conformation requires 100 μsec (Sawicki and Gibson 1976; Marden et al. 1986). Thus ligand rebinding may occur on the same timescale as the R to T transition; at sufficiently high ligand concentrations, the ligands may recombine to the rapid R-state before significant conversion. Samples are often equilibrated <0.1-atm CO to decrease the on-rates and allow a full conversion to the T-state.

Increasing the CO concentration caused a decrease in the relative amplitude of the slow phase of CO recombination to Hb1a (Fig. 5 ▶). As for unmodified HbA, this would indicate an R to T transition after photolysis. For Hb 1b there was little change; either the R to T conversion is rapid, or a fraction of the tetramers is already in the T-state before photolysis. To distinguish between these two possibilities, one needs an allosteric probe of the fully liganded form.

Fig. 5.

CO recombination to hemoglobin (Hb) 1a for samples equilibrated with 0.1 (with data points, upper time axis) and 1 atm CO, at pH 7, 37 °C. The kinetics for the samples equilibrated <1-atm CO (without data points, lower time axis) show a lower fraction of the slow (T-state) phase, indicating that CO rebinding to the preflash liganded (R-state) is competitive with the allosteric transition.

Fluorescent marker

One such probe is a fluorescent analog of 2,3-diphosphoglycerate: hydroxy-pyrenetrisulfonate (HPT; MacQuarrie and Gibson 1972^; Marden et al., 1986). Fluorescent studies with HPT did not show the usual quenching by Hb, indicating a lower affinity of the cross-linked Hbs for the effector. This explains why these cross-linked Hbs did not show a DPG effect on oxygen binding (Abraham et al. 1995; Boyiri et al. 1995).

Geminate yield

A second probe is the fraction geminate recombination. Usually Hb shows ∼50% geminate recombination for CO at 25°C. The fraction geminate depends on the relative rates for rebinding versus escape. Generally, the slower the rebinding rate, the higher the fraction bimolecular kinetics; MbCO, for example, has a lower on-rate (than R-state Hb) and shows <3% geminate phase. Similarly studies of singly liganded Hb have indicated a low geminate fraction for T-state Hb (Sawicki and Gibson 1979; Marden et al. 1987; Murray et al. 1988); triply NO Hb with IHP also displays little geminate phase for CO binding to the fourth site (Kiger et al. 1993). Thus the fraction bimolecular phase, an easily measured value, can also be used as a probe to estimate the percent of each allosteric state. Note that a difference in the R-state rates will also influence this fraction. For example, addition of IHP to Hb causes a change in both the R and T rates, and the R' rate for CO is lower than for the native R-state, leading to a lower geminate fraction.

Oxygen off-rate

The rate of ligand dissociation from the fully liganded form is also a test of the protein conformation. For these measurements, oxygen is the best choice of ligand. Just as CO displays a large difference in association rates for the two allosteric conformations, this difference is expressed mainly in the off-rates for oxygen. A fraction of T-state Hb would appear as a faster phase of the oxygen dissociation kinetics; if the T-state phase was too fast for detection, there would be a decrease in the amplitude of the slower phase. The kinetics for the dissociation of oxygen for Hb 1d, which has the longest cross-linking molecule in the bis-aldehyde series (Fig. 1 ▶), were similar to those for HbA.

These results indicate an R-like conformation for the fully liganded form. The overall bimolecular amplitudes are similar to that for HbA and increases with temperature. Small differences in the bimolecular yield could be attributed to a modified R-state, rather than a fraction T-state. The fully liganded form of the cross-linked Hbs could then be considered as an R'-state with properties similar to the R-state of HbA.

The variation of the relative amounts of rapid and slow bimolecular kinetics versus the fraction dissociation represents a deviation from the predictions by the Monod, Wyman, Changeux (MWC) model, which requires the same shift in allosteric equilibrium (c = K_R/K_T) for each ligand. To obtain T-like recombination kinetics to the triply liganded form (Fig. 4 ▶), the allosteric equilibrium L3 = T3/R3 must be at least 10. This would imply a significant fraction of fully liganded form in the T-state: L4 = L3 * c > 0.1. Yet we did not see evidence for a significant fraction T4, based on the bimolecular yield for photodissociation.

Oxygen on-rate

Recombination kinetics of oxygen after photolysis are shown in Figure 6 ▶. Because the yield for the bimolecular phase is low (<5%) with oxygen as ligand, the signals are small and the main photoproduct is the triply liganded tetramer (as for HbCO at low photolysis levels). Samples were equilibrated under air (Fig. 6 ▶); similar shifts in the kinetics were observed for samples under 1 atm oxygen to obtain a higher saturation level. Although most modulators show little effect on ligand binding to the "relaxed" conformation, certain cross-linked Hbs show a shift in the kinetics. These kinetic differences may represent small changes in tertiary structure leading to modified (R' and T') forms of both allosteric states.

Fig. 6.

Oxygen rebinding after photodissociation to (air equilibrated) modified hemoglobins (Hbs). As the quantum yield is low for oxygen, <5% of the hemes are without ligand at the start of the bimolecular phase. The recombination thus probes the rate for the binding of the fourth ligand.

Stability of tetramer

At low protein concentrations, the photolysis kinetics of unmodified HbA show a lower fraction of the slow phase. This is due to an enhanced dimer fraction, because dimers show only the rapid R-like phase. Thus the fraction slow versus protein concentration can be used to determine the dimer–tetramer equilibrium coefficient (Khaleque and Sawicki 1988). At pH 7, concentrations below 1 μM of HbA are necessary to observe a significant change. We studied a few of the cross-linked forms at 0.5 μM (on a heme basis) and observed no change in the shape of the kinetics.

Dimer exchange

Liganded hemoglobin tetramers normally dissociate readily into dimers, whereas the deoxy form is predominantly tetrameric (Thomas and Edelstein 1972). The rate coefficient for dimerization of liganded tetramers is on the order of 1 sec (Nagel and Gibson 1971), and mixing HbCO with HbCN yields stoichiometric amounts of the hybrids [dimer-CO]/[dimer-CN]. The CO ligands (but not CN) can be photodissociated from these hybrids to study CO rebinding to the asymmetric form [dimer-deoxy]/[dimer-CN] (Marden et al. 1996). The CO recombination kinetics to the cross-linked Hb, with and without metHbCN, are shown in Figure 7 ▶. Whereas HbA shows a decrease in slow phase when metHbCN is added, the cross-linked Hb shows little change.

Fig. 7.

CO recombination kinetics for a mixture of cross-linked hemoglobin (Hb) 1c with normal metHbA-CN. Although unmodified HbA shows an interaction with metHb-CN via dimer exchange, the cross-linked Hb does not show evidence for the dimer exchange. Curves with data points show the kinetics after addition of metHb-CN.

Autoxidation rate

The autoxidation rate of HbA is 16-fold higher at low concentration than under the tetrameric form (Kiger et al. 1993). Autoxidation studies at low concentration of the crosslinked Hbs showed only a slight acceleration. Addition of haptoglobin did not increase the oxidation rate, again suggesting that dimers are not free.

Discussion

The kinetic results show a large range in the relative proportions of fast and slow bimolecular recombination (Fig. 3 ▶). The intrinsic rates also change but remain within a factor of 2 relative to HbA, whereas the allosteric equilibrium changes by over two orders of magnitude.

Simulations with a two-state model did not provide satisfactory description of the entire range of data versus fraction photodissociation. If a sample shows a significant amount of T-state for the triply liganded form (as observed at low photolysis levels), then one would expect pure T-state conformation for tetramers with ≤2 ligands. Yet there was still a persistent rapid fraction even at the higher photolysis levels. If the two-state parameters were adjusted to fit curves at low dissociation levels, then the experimental curves at high dissociation had too much rapid phase relative to simulations. An excess of rapid phase at high dissociation levels is often attributed to a dimer contribution for normal samples; a fraction without the cross-link or improperly linked could explain this result. However, an independent fraction of high affinity protein would be evident in the low saturation region of oxygen equilibrium curves; this was not observed. In any case, simulations versus laser energy did not provide consistent results for the relative amount of slow phase.

Dimer exchange

There was no effect of protein concentration, to values as low as 0.5 μM in heme, on the CO rebinding kinetics. This provides evidence that the cross-link stabilizes the tetrameric form.

Another test of the tetramer stability is to mix the Hb with an excess of cyano-metHbA (Marden et al. 1996). For HbA-CO, exchange of dimers produces [dimer-CO/dimer-CN] hybrids, which have quite different photolysis properties from Hb(CO)₄. Because CN is not photodissociable, the photoproducts of the hybrids do not contain deoxy or singly liganded tetramers. For unmodified HbA, the CO-rebinding kinetics show much less slow phase relative to HbCO. No changes after mixing were observed for the cross-linked Hbs 1c and 1a, indicating no dimer exchange. Little change might be expected for Hb 1c because even the triply liganded forms show a large fraction of slow phase (Table 1).

DPG effect

The addition of DPG (or IHP) made little difference in the oxygen or CO-binding properties for these cross-linked Hbs. Because the allosteric equilibrium is already shifted by the cross-link, DPG may bind but with little additional effect. Alternatively, the affinity for DPG may be reduced. Direct binding studies of the effector are difficult, but a fluorescent analog HPT allowed such a test. Although the fluorescence decreases for HPT binding to deoxy HbA, the decrease was not observed with Hb1c under the same conditions. This indicates that the DPG site is blocked or perturbed by the cross-link.

Evidence for a T4 state

In some cases the allosteric equilibrium is shifted so far that the Hb remains in the T-state conformation even with three ligands bound. One can then ask whether the fully liganded tetramer is in the R or T state or a slightly modified R' or T' state. Strong effectors such as IHP induce a large shift in the allosteric equilibrium, with modified properties of the deoxy and oxy forms, but the fully liganded form remains in an R-like conformation.

Ligand-binding models

The cross-linked series of Hbs show evidence for only two allosteric conformations. For the forms with a large shift in the allosteric equilibrium, mainly the T-like properties are observed in the ligand-binding kinetics, and it is not clear whether the fully liganded forms have a significant T-state contribution. In this case ligand dissociation rates are a more sensitive test; the oxygen off-rate from Hb 1c was similar to normal Hb, whereas the T-state rate is calculated to be ∼30 times higher. The fraction of geminate recombination is also sensitive to the allosteric state before photodissociation. A higher bimolecular yield was observed, but that could be attributed to a different R' association rate. For these compounds, there is no evidence for a stable T4 state.

The monoaldehyde allosteric modifiers also bind to α Val 1 but can have opposite effects on the allosteric equilibrium. Those that produced low affinity Hbs were found to bridge the dimer–dimer α subunits with a hydrogen bond instead of a covalent cross-link as formed by the bis-aldehydes. The replacement of a covalent cross-link with a hydrogen bond produces a much lower effect on the allosteric equilibrium. Although DM-HbA also forms a Schiff-base interaction with Val 1α, as do the mono-and bis-aldehydes, it does not form an interaction with the α or β chains of the opposite dimer and therefore destabilizes the T-conformation, producing an opposite shift in the allosteric equilibrium (Abraham et al. 1995).

Conclusions

The previous studies showed that the cross-linking series (1a–1c) produce a significant decrease in Hb oxygen affinity (Boyiri et al. 1995). The conclusion drawn in that work was that the chain length acts as a molecular ratchet so that the shorter the chain, the tighter the constraint on the T-state and the greater the decrease in the oxygen affinity (series 1b–1d). The one exception was the cross-linking agent 1a with only one methylene that is not quite long enough to position the second aldehyde for cross-linking to Lys 99α of the opposite subunit. In this study we obtained direct evidence from the CO bimolecular kinetics for the shift in the allosteric equilibrium; the variable shift in the fraction of T-like kinetics show the molecular ratchet hypothesis.

The studies of mixtures of cross-linked Hb-CO with Hb-CN did not show evidence of dimer exchange, unlike normal HbA, confirming the stabilization of the tetrameric form. Only two CO on-rates (or two oxygen affinities for the equilibrium measurements) were observed; thus one can maintain a two-state framework for describing the results. Changes are observed in the ligand affinities or binding rates, but the variation remains within a range of a factor of 2. Because these differences are small compared to the factor-of-100 difference between the R and T states, we refer to these minor modifications as R' or T' states. The dominant effect of the cross-link is a change in the allosteric equilibrium, because the switchover point varies from about 1 to 3, corresponding to a change in the allosteric equilibrium of over a factor of 10,000. The shift due to the cross-link is apparently not additive with the usual effector DPG; in most cases there was little additional change due to addition of DPG or IHP. However, the effects of pH and NaCl remain present for the cross-linked hemoglobins. This would indicate that one can eliminate dimers and regulate the oxygen affinity by a proper design of the cross-link.

Materials and methods

Cross-linked Hbs were obtained by incubating HbA with the various cross-linking agents for 24 h at pH 7, followed by addition of sodium cyanoborohydride to reduce the Schiff base, as previously described (Wireko and Abraham 1991; Boyiri et al. 1995). The cross-linked Hb samples were crystallized in the deoxy form and the crystals were kept refrigerated (4°C) until use. The three-dimensional structure was previously determined (Abraham et al. 1995; Boyiri et al. 1995) and confirms the binding sites on the α chains (Fig. 2 ▶). Crystals were dissolved in the appropriate buffer for the functional studies.

Absorption spectra were measured with an SLM-Aminco (DW2000) spectrophotometer. Spectra for the oxy, deoxy, CO, and cyano-metHb forms were normal; we assumed the same absorption coefficients as for the control HbA to determine the fraction dissociation.

Fluorescence measurements were made with an SLM-Aminco 8000 spectrometer. HPT (8-hydroxy-1,3,6-pyrenetrisulfonate, Kodak) was used as the fluorescent analog of DPG, at a concentration of 10 μM in 10 mM bis-Tris buffer at pH 7. Emission spectra were measured in the visible region with excitation at 331 nm; excitation and emission slits were at 4 nm and 2 nm, respectively.

Kinetics

CO-and oxygen-rebinding rates were measured after photolysis by 10-nsec laser (Quantel) pulses at 532 nm. The CO bimolecular kinetics provide information on the association on-rates for the R and T conformations, as well as the allosteric equilibrium. Measurements were made at different photolysis levels for samples equilibrated under air, 1 atm oxygen, 0.1 or 1 atm CO. The oxygen dissociation rates were measured by stopped flow; Hb samples under 1 atm oxygen, to insure maximum saturation, were mixed with a 5-mM solution of the oxygen scavenger Na-dithionite.