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Originally published In Press as doi:10.1074/jbc.M307515200 on August 17, 2003

J. Biol. Chem., Vol. 278, Issue 44, 42769-42773, October 31, 2003
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Allosteric Effect of Water in Fish and Human Hemoglobins*

Christian Hundahl, Angela Fago, Hans Malte, and Roy E. Weber{ddagger}

From the Department of Zoophysiology, Institute of Biological Sciences, C. F. Møllers Alle 131, University of Aarhus, DK-8000 Aarhus C, Denmark

Received for publication, July 14, 2003 , and in revised form, August 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prompted by the reported lack of solvation effects on the oxygen affinity of fish (trout I) hemoglobin that questioned allosteric water binding in human hemoglobin A (Bellelli, A., Brancaccio, A., and Brunori, M. (1993) J. Biol. Chem. 268, 4742–4744), we have investigated solvation effects in fish and human hemoglobins by means of the osmotic stress method and allosteric analysis. In contrast to the earlier report, we demonstrate that water potential does affect oxygen affinity of trout hemoglobin I in the presence of inert solutes like betaine. Moreover, we show that upon oxygenation electrophoretically anodic hemoglobin from trout and eel bind a similar number of water molecules as does human hemoglobin A, whereas the cathodic hemoglobins of trout and eel bind smaller, but mutually similar, numbers of water molecules. Addition of cofactors strongly increases the number of water molecules bound to eel hemoglobin A (as in human hemoglobin) but only weakly affects water binding to eel hemoglobin C.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well established that changes in water activity regulate oxygen binding in adult human hemoglobin (HbA).1 Upon oxygen binding, human HbA becomes more hydrated, taking up 60–70 water molecules; an increase in water activity thus increases oxygen affinity (1–4). The greater hydration of the R ("relaxed" oxygenated) state of HbA compared with the T ("tense" deoxygenated) state is consistent with the (500–800 Å2) greater surface exposed to the solvent in the R state (5, 6).

How do other tetrameric Hbs respond to changes in water activity? Despite extensive data on the water sensitivity of human HbA, very little is known about how water affects oxygen binding in other tetrameric vertebrate Hbs. Fish Hbs that show a remarkably broad range of oxygen binding properties and allosteric effects and are well characterized from functional and structural points of view (7, 8) are excellent candidates for such analysis. In contrast to mammals that often have only one major Hb component, fish commonly have several isoHbs with marked functional differentiation, indicating an in vivo division of labor to ensure adequate O2 loading and unloading under various physiological and environmental conditions (7). Fish Hbs fall into two major categories (9) based on their electrophoretical mobility at alkaline pH: 1) anodic Hbs found in all fish, whose oxygen affinities are strongly decreased by heterotropic ligands such as protons (Bohr and Root effects) and organic phosphates, and 2) cathodic Hbs encountered in eels, salmonids, and catfish, which show no or little Bohr and highly variable phosphate effects. Because of their higher O2 affinities and lower pH sensitivities, the cathodic Hbs are well suited for O2 transport under hypoxic, hypercapnic, or acidotic conditions (9). A feature distinguishing anodic fish Hbs from other pH-sensitive tetrameric Hbs, such as human HbA, is the Root effect, where drastic reductions in oxygen-carrying capacity and cooperativity at acidic pH marks stabilization of the low affinity T state with loss of the oxygen-driven, cooperative T-R transition (10).

The purpose of the present study is to analyze the water effect in various Hbs with different oxygen affinities and sensitivities to allosteric effectors, including human HbA and the cathodic and anodic Hbs from trout (Oncorhyncus mykiss) and eel (Anguilla anguilla). Whereas the anodic Hbs from trout and eel are functionally similar, both showing Bohr and Root effects (11–15), pH-insensitive cathodic fish Hbs exhibit highly variable allosteric properties. Whereas trout cathodic HbI is insensitive to organic phosphates (11, 13), oxygen binding in cathodic eel HbC shows high organic phosphate sensitivity (11, 16).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human HbA and cathodic and anodic Hbs from eel and trout were stripped from organic phosphates, and purified as described (9, 17, 18). All preparative procedures were carried out at 0–5 °C. The Hb solutions were stored at –80 °C in aliquots that were thawed immediately before oxygen binding studies. Water activity was varied by addition of inert solutes (which do not interact with the protein surface and therefore only have an indirect effect on oxygen affinity through changes in the water activity). In this "osmotic stress" method (1, 19), changes in the Hb oxygen affinity are related to changes in water activity that can be converted to changes in protein hydration by use of linkage equations.

To Hb solutions (0.1 mM heme) at pH 7.3 in 0.1 M HEPES buffer, appropriate aliquots of standard sucrose, betaine, or glycine stock solutions were added in order to achieve different water activities (aw). Sucrose was used to replicate the conditions adopted by Bellelli et al. (18) on trout HbI. Betaine was chosen because it was found to be preferentially excluded from protein surfaces (20). Glycine is conveniently used as inert solute in osmotic stress studies because it does not show significant interaction with protein (including Hb) surfaces (2).

O2 equilibria of 4-µl Hb subsamples were recorded using a modified gas diffusion chamber fed by cascaded Wösthoff gas-mixing pumps that produce stepwise increases in O2 tension by mixing air or O2 with ultrapure (>99.998%) N2 while absorbance at 436 nm was recorded continuously (21). A BMS 2 MK 2 thermostated microelectrode (Radiometer, Copenhagen, Denmark) was used to measure pH in 100-µl subsamples. All equilibria measurements were carried out at 15 °C. O2 affinity (P50, half-saturation O2 tension) and the cooperativity (Hill) coefficient at half saturation (n50) were interpolated from Hill plots [log (Y/(1–Y) versus log PO2, where Y is the fractional saturation and PO2 is the O2 tension] constructed from data points between 30 and 70% saturation. Osmolalities were measured in 150-µl samples on a Semi-Micro Osmometer (Knauer, Kiel, Germany), and water activities were calculated according to Ref. 2 as shown in Equation 1

(Eq. 1)
where Osm is the solution osmolality (mol Kg–1) and Mw is the molality of pure water (55.56 mol Kg–1).

To analyze the effect of water as a single heterotropic ligand on oxygenation, we used the Wyman linkage equation (1, 22, 23) as shown in Equation 2

(Eq. 2)
where aw is the activity of water. The slope of the linkage plot ln(P50) versus ln(aw) gives the apparent difference in number of water molecules bound to the oxy and deoxy structures of Hb, {Delta}nw. This analysis is based on the assumption that the activity of only a single heterotropic ligand, in this case water, varies. If the activity of more than one heterotropic ligand (e.g. solute and water) varies, the number of oxygen-linked water molecules will be overestimated.

An extended linkage equation based on the Gibbs-Duhem relationship allows quantification of the separate contributions of solute and water to the regulation of O2 binding. Starting from the Gibbs-Duhem relation one can show (2), as in Equation 3, that

(Eq. 3)
where ms and mw are solute and water molality, respectively, and Pm is the median oxygen tension. For a reasonably symmetric oxygen equilibrium curve, Pm can be replaced by P50. and realizing that dln(as) = dln(ms) Equation 3 can be integrated to yield, as shown in Equation 4,

(Eq. 4)
where , is P50 at 1000 mosM. The data were plotted as function of ln(aw) or ln(ms) and fitted by least-squares procedures to either the Wyman (Equation 2) or the Gibbs-Duhem (Equation 4) equations in order to obtain {Delta}nw and {Delta}ns, the apparent number of oxygen-linked water and solute molecules, respectively. For detailed analysis of the allosteric interactions, oxygen equilibrium measurements were carried out at extreme (low and high) fractional saturations and fitted according to the Monod-Wyman-Changeux model (24) as shown in Equation 5

(Eq. 5)
where L is the allosteric constant in the absence of ligand defined as [T0]/[R0], KT and KR are the intrinsic ligand affinities of the T and R states, respectively. The parameters of the equation were estimated from non-linear least-squares curve fitting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In human HbA and the anodic trout and eel Hbs, an increase in water activity increases oxygen affinity (Fig. 1), as evident from the slopes of plots of versus ln(aw) (Fig. 1A) and versus ln(Osm) (Fig. 1B). The effect of changes of water and solute activity is identical for all three Hbs, and analysis of the data according to the Wyman and Gibbs-Duhem equations shows binding of 42–43 additional water molecules in the T-R transition (Table I). Moreover, the different solutes tested have a similar effect on oxygen binding independent of their physical and chemical properties (Fig. 1). This indicates that they only affect oxygen affinity indirectly through changes in water activity, which validates their use in osmotic stress experiments. Increasing water activity also increases O2 affinity of the cathodic Hbs (trout HbI and eel HbC) to the same extent and independently of the use of betaine or glycine (Fig. 2) but to a lesser degree than in the anodic Hbs. Curve fitting of the data shows binding of 17–31 water molecules bound upon oxygenation (Table I), reflecting lower water sensitivity than in human HbA and the anodic fish Hbs. However, the addition of sucrose does not affect the oxygen affinity of trout HbI as previously reported (18).



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  		FIG. 1.
The relative shift in (), , related to water activity, ln(aw), fitted with the Wyman equation (A), and to solute osmolality, ln(Osm), fitted with the Gibbs-Duhem equation (B), for human HbA (circles), anodic trout HbIV (triangles), and anodic eel HbA (squares) at pH 7.3, in the presence of betaine (closed symbols), glycine (open circles and squares), and sucrose (open triangles).

 

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  		TABLE I
Linkage parameters for O2 binding and heterotropic water and solute binding in investigated hemoglobins

Change in the number of water ({Delta}nw) and solute molecules ({Delta}ns) bound per tetramer in the transition from fully deoxygenated to fully oxygenated Hb in different solvents as estimated from Equations 2 and 3. pH 7.3; [GTP] = 0.05 mM; [KCl] = 0.1 M.

 


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  		FIG. 2.
The relative shift in P50, , related to water activity, ln(aw), fitted with the Wyman equation (A), and to solute osmolality, ln(Osm), fitted with the Gibbs-Duhem equation (B), for trout HbI (circles) and eel HbC (triangles) at pH 7.3, in the presence of betaine (Bet), sucrose, and glycine (Gly) as indicated.

 



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  		FIG. 4.
The relative shift in P50, , related to water activity, ln(aw), fitted with the Wyman equation (A), and to solute osmolality, ln(Osm), fitted with the Gibbs-Duhem equation (B), for eel HbC at pH 7.3 in the absence (open circles) and presence (solid circles) of 0.05 mM GTP and 0.1 M KCl.

 


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  		FIG. 3.
The relative shift in P50, , related to water activity, ln(aw), fitted with the Wyman equation (A), and to solute osmolality, ln(Osm), fitted with the Gibbs-Duhem equation (B), for eel HbA at pH 7.3 in the absence (circles) and presence (triangles) of 0.05 mM GTP and 0.1 M KCl.

 

The oxygen-affinity modulators affect water binding to eel HbA (Fig. 3) in a pronounced manner. Addition of KCl and GTP increases the number of water molecules involved in the deoxy to oxy transition from 42–43 to 118–129 per tetramer (Table I).

An analogous effect of KCl and GTP was not seen in cathodic eel HbC (Fig. 4; Table I). Eel cathodic HbC is clearly less sensitive to changes in water activity than eel anodic HbA, both in the presence and absence of cofactors. However, for all Hbs investigated here oxygen affinities increase with increasing water chemical potential.

Extended Hill plots derived from precise oxygen equilibrium measurements over a wide saturation range were carried out on human HbA, eel HbA, and HbC in the absence and presence of 1 M betaine to obtain insight into the allosteric control mechanism underlying the effect of water activity. Decreasing water activity affects human HbA and anodic and cathodic eel Hbs in different ways (Fig. 5, A–C). Whereas an increased solute level decreases only KT in human HbA (Fig. 5A), it decreases both KT and KR in eel HbA and HbC, decreasing L more strongly in HbC than in HbA (Fig. 5, B and C; Table II).



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  		FIG. 5.
Extended Hill plots of human HbA (A), eel HbA (B), and eel HbC (C) in the presence (filled circles) and absence (open circles) of 1 M betaine. Inserted panels show the values of KT (filled circles), KR (open circles) and L (filled triangles) as a function of water activity, ln(aw)·10–2.

 

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  		TABLE II
MWC and derived parameters for human HbA, eel HbA, and eel HbC at 15 °C in the absence and presence of 1 M betaine

P50 and Pm (half-saturation and median O2 tension values in mm Hg); KT, KR (O2 association constants of low-affinity (tense) and high-affinity (relaxed) states, respectively) and S.E. values (in mm Hg–1); n50 and nmax (cooperativity coefficients at half-saturation and maximal values, respectively); L, allosteric constant ([T]/[R] in the absence of O2); {Delta}G (free energy of cooperativity in kJ/mol). The Monod-Wyman-Changeux (MWC) model was fitted according to Equation 5.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found only slight differences in the number of oxygen-linked water molecules when fitting the Wyman linkage equation and the Gibbs-Duhem equation, respectively. Such differences have been previously reported (2) and reflect the intrinsic assumption of the Wyman equation in osmotic stress experiments that oxygen affinity responds only to water activity changes and that solutes do not undergo functionally significant interaction with the protein surface. This assumption is supported by the finding that the number of oxygenation-linked, bound solute molecules estimated with the Gibbs-Duhem equation is close to zero and independent of the chemical nature, size, and charge of the solutes. Accordingly, the closer {Delta}ns is to zero, the better the agreement between the {Delta}nw values calculated using the two equations (Table I). However, the agreement between {Delta}nw values calculated by the two equations may not be uniquely related to the absence of solute binding, as indicated by the similar values for eel HbA, which shows apparent oxygen-linked solute binding in the presence of GTP + KCl (Table I).

Only ~25 additional water molecules bind to human HbA during the deoxy to oxy transition in the absence of anions, in contrast to ~72 molecules in the presence of saturating levels of Cl– or 2,3-diphosphoglycerate (3). The discrepancy of 45–50 water molecules was ascribed either to local dehydration of the anion binding site upon complex formation or to global change in the protein conformation induced by anion binding, although a release of 45–50 water molecules was assumed to be too large an effect to be attributed only to the reorganization of bound water molecules at the phosphates binding site (3).

Our value of 42–43 water molecules that are bound to human HbA during oxygenation is intermediate between the values of ~25 molecules found for stripped, deionized HbA in weak (10 mM Hepes) buffer (3) and ~60–70 found for HbA (1) in 0.1 M NaCl and 0.05 M Tris buffer, suggesting sensitivity to different buffer conditions. We here show that the physiological effectors GTP and Cl– increase the number of water molecules bound to eel HbA from 40–43 in stripped Hb to 118–129 (Table I). The structural basis for this increase in hydration is not known or whether the increase can be related to the strong phosphate and pH effects observed in anodic fish Hbs.

The cathodic fish Hbs studied here (trout HbI and eel HbC) analogously respond to an increase in water activity with an increased oxygen binding, indicating preferential binding of water molecules to the R state. These Hbs bind a mutually similar but smaller number of water molecules than the anodic Hbs during the deoxy to oxy transition (~17–31, when betaine is used as solute; Table I) despite marked differences in their sensitivity to organic phosphates, which is lacking in trout HbI and pronounced in eel HbC (9, 25).

Bellelli et al. (18) have previously shown that addition of sucrose to trout HbI solutions had no effect on oxygen binding, suggesting that trout HbI may be insensitive to changes in water activity. To test this, we examined the effects of sucrose and of betaine, which is the solute least likely among a range of tested solutes to interact with protein surfaces (20). In agreement with the observation of Bellelli et al. (18), trout HbI showed almost no response to varying sucrose concentrations but did show similar responses as the other tetrameric Hbs investigated in the presence of betaine, binding ~17–31 additional water molecules upon oxygenation (Fig. 2; Table I). The results obtained with betaine may thus represent a general response to changes in water activity among tetrameric Hbs. A possible explanation for the lack of a water effect in trout HbI in the presence of sucrose may be that sucrose has a 2-fold effect on this Hb, one on water activity (stabilizing the R state) and one on the dielectric constant of the medium (stabilizing the T state; Ref. 18). These two effects may act in opposite directions and obliterate each other. This can be compared with the Cl– effect on human HbA that involves both direct interaction with the protein and an indirect effect exerted through a change in water activity (26, 27). Sucrose and Cl– would thus be poor choices for investigating osmotic stress in trout HbI or human HbA, respectively. In contrast to trout HbI, sucrose and betaine have superimposable effects on trout HbIV and are in this case inert solutes. Sucrose, glycerol, and glucose also have superimposable effects on the oxygen affinity of the clam Scapharca inaequivalvis HbI (28), supporting the view that the apparent sucrose insensitivity in trout HbI is due to specific properties of this Hb molecule. The highly consistent results obtained with betaine in the proteins investigated here confirm the inference of Courtenay et al. (20) that betaine may be the most appropriate solute in osmotic stress experiments and in protein hydration studies.

In studies on solvation effects in proteins, differences in the size of the solvent-accessible area between conformational states are determining factors (1). An interesting feature of trout HbI is that compared with human HbA it has a larger buried surface area in the R state than in the T state (17). Nevertheless it binds water molecules upon oxygenation, albeit to a lesser extent than human HbA. This indicates that factors other than surface accessible area influences protein hydration. One possibility is that upon oxygenation of trout HbI the ratio of polar to hydrophobic amino acid surface residues increases even though the overall solvent-accessible area decreases.

Taken together, human and anodic and cathodic fish Hbs show similar overall sensitivities to changes in water activity despite the diversity in their pH and phosphate effects. This suggests that the amino acid residues responsible for allosteric effects, such as the Bohr effect and phosphate and anion sensitivities, do not necessarily contribute to water sensitivity. This seems logical because water sensitivity may be expected to rely on the presence/absence of hydrophilic residues on the protein surface, whereas the requirements for Bohr groups are more stringent and relate to their precise spatial distribution and changes in proton binding affinity in response to ligand binding. In the highly phosphate-sensitive eel HbC, addition of GTP and KCl does not alter the allosteric water binding, suggesting that the central cavity at the phosphate-binding site does not contribute to differential hydration between T and R states (Fig. 4). This is the opposite of anodic eel HbA where addition of allosteric cofactors increases the number of water molecules bound upon oxygenation almost 3-fold (Fig. 3; Table I), resembling the situation in human HbA discussed above. As also concluded by Colombo and Seixas (3), it seems unlikely that this strong interplay between allosteric anions and water binding should reflect reorganization of bound water molecules at the anion binding sites alone and may reflect more radical conformational changes. Clearly, some basic structural differences seem to exist in phosphate-induced conformational changes between eel Hbs C and A.

In conclusion, we show that all the tetrameric Hbs investigated here respond to an increase in water activity by stabilizing the R state conformation and that the number of oxygen-linked water molecules is similar among anodic Hbs (human HbA, eel HbA, and trout HbIV) and among cathodic ones (eel HbC and trout HbI), ~43 and ~17–31, respectively. The only data on fish Hbs that hitherto were available (showing absence of hydration effects on trout HbI, Ref. 18) thus cannot be considered representative of fish Hbs and may have been influenced by the specific experimental conditions and solute choice.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 45-8942-2599; Fax: 45-8619-4186; E-mail: roy.weber{at}biology.au.dk.

1 The abbreviations used are: Hb, hemoglobin; R, relaxed; T, tense. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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