Functional modulation by lactate of myoglobin. A monomeric allosteric hemoprotein.

The effect of lactate on O2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O2 affinity (i.e. an increase of P50) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O2 affinity appears to differ for sperm whale and horse heart Mb, δlogP50 being ≈1.0 and ≈0.4, respectively. From the kinetic viewpoint, the variation of O2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.

The effect of lactate on O 2 binding properties of sperm whale and horse heart myoglobins (Mb) has been investigated at moderately acid pH (i.e. pH 6.5, a condition which may be achieved in vivo under a physical effort). Addition of lactate brings about a decrease of O 2 affinity (i.e. an increase of P 50 ) in sperm whale and horse heart myoglobins. Accordingly, lactate shows a different affinity for the deoxygenated and oxygenated form, behaving as a heterotropic modulator. The lactate effect on O 2 affinity appears to differ for sperm whale and horse heart Mb, ␦logP 50 being Ϸ1.0 and Ϸ0.4, respectively. From the kinetic viewpoint, the variation of O 2 affinity for both myoglobins can be attributed mainly to a decrease of the kinetic association rate constant for ligand binding.
Myoglobin (Mb) 1 is a monomeric hemoprotein present in muscle cells of most vertebrates and invertebrates which reversibly binds O 2 with a fairly high affinity (1). Since up to now its function has not been reported to be modulated by environmental conditions, Mb has been thought to display two different and interconnected roles: (i) to be a reserve supply of O 2 and (ii) to facilitate the oxygen flux within a myocyte (2)(3)(4).
It has been reported recently that Mb shows ligand-linked tertiary conformational changes (5,6), suggesting the possibility that the equilibrium between different structural arrangements of the molecule may be affected by non-heme ligands. In the present study, we show that the functional properties of sperm whale and horse Mbs indeed are influenced by lactate, an obligatory product of glycolysis under anaerobic conditions, which appears to play a modulatory role for Mb function, as much as organic phosphates and/or protons (not effective on Mb) influence the function of hemoglobin (7). Lowering of the O 2 affinity of Mb by lactate may have relevant physiological consequences, since during a transient tissue hypoxia (such as that which may be induced by a physical effort or diving) the increase of lactate concentration may induce Mb to release O 2 , thus facilitating its diffusion to mitochondria and helping to keep constant the turnover rate of oxidative phosphorylation.

EXPERIMENTAL PROCEDURES
Sperm whale Mb, horse heart Mb, and MES were obtained from Sigma. Lactate has been purchased as L-(ϩ)-lactic acid from Fluka Chemie AG (Buchs, CH) and used without further purification. All chemicals were of reagent grade and used without further purification.
The deoxygenated derivative of sperm whale and horse heart Mb has been obtained: (i) by reducing the commercially available ferric form with minimal amounts of sodium dithionite and (ii) by carefully degassing the oxygenated form. The oxyMb derivative of sperm whale and horse heart has been prepared by eluting the deoxygenated protein solution through a Sephadex G-25 gel filtration column to remove the excess of sodium dithionite; only the first elution portion was collected and used immediately (1).
Values of the dissociation equilibrium constant (K O obs ϭ P 50 ⅐2.7 ϫ 10 Ϫ4 /152) for oxygen binding to sperm whale and horse heart Mb, in the absence and in the presence of lactate (from 0.005 to 0.1 M) were obtained spectrophotometrically by the tonometric method, employing a large volume tonometer (Ϸ300 ml) equipped with a cuvette to acquire absorption spectra as a function of oxygen pressure (8). The low solubility of lactate precluded the possibility of raising its concentration above 0.2 M in an accurate fashion.
Values of the dissociation equilibrium constant for lactate binding to the oxygenated derivative of sperm whale and horse heart Mb were obtained spectrophotometrically by following the decrease of the absorption spectrum of MbO 2 at 418 nm upon ligand binding. No spectral changes have been detected for the interaction of lactate with deoxyMb.
The second order rate constant for oxygen binding to horse heart Mb, in the absence and in the presence (0.2 M) of lactate, was calculated from the dependence of the apparent pseudo-first order rate constant for MbO 2 complex formation (i.e. k obs ) on ligand concentration. Values of k obs were determined by mixing deoxygenated horse heart Mb (obtained by carefully degassing the Mb oxygenated derivative in the absence of sodium dithionite) with a buffered solution containing the desired amount of oxygen (1). Values of the first order rate constant for O 2 dissociation from sperm whale and horse heart Mb were obtained by mixing either (i) the oxygenated derivative of Mb with sodium dithionite or (ii) the deoxygenated form of Mb, in the presence of sodium dithionite, with a buffered solution saturated with oxygen (i.e. the O 2 pulse experiment (9)).
Kinetic experiments have been undertaken at the Department of Biochemical Sciences "Alessandro Rossi Fanelli" of the University of Rome "La Sapienza," employing a Gibson-Durrum stopped-flow with a 2-cm path length observation cell interfaced to a desk-top computer for fast data acquisition (On Line Instrument Service, Jefferson, GA).
All spectrophotometric measurements were performed with a Varian Cary 219 spectrophotometer. All experiments have been performed in 0.1 M MES, at pH 6.5 and 20°C. Fig. 1 shows the effect of lactate concentration on the O 2 binding properties of sperm whale and horse heart Mb, at moderately acidic pH values (i.e. pH 6.5 in 0.1 M MES), a condition which may be achieved in vivo under a physical effort (10). For both Mbs, a significant decrease of O 2 affinity (i.e. an increase of P 50 and K O obs values) can be observed (see Fig. 1). However, the effect of lactate is different for the two hemopro-* This work was supported by funds of Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica of Italy (MURST 40%) and of Consiglio Nazionale delle Ricerche of Italy (CNR). 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.

RESULTS AND DISCUSSION
‡ ‡ To whom correspondence should be addressed. Tel.: 39-737-40706; Fax: 39-737-636216. 1 The abbreviations used are: Mb, myoglobin; MES, 4-morpholineethanesulfonic acid. teins, being larger for sperm whale Mb than for horse heart Mb (␦logP 50 Ϸ1 and Ϸ0.4, respectively). The effect of 5 mM lactate, a concentration easily attainable inside myocytes during hypoxia (11,12), indicates that this ligand may exert a physiologically relevant effect on the O 2 binding properties of Mb under hypoxic conditions in vivo (see Fig. 1).
The effect of lactate on the Mb functional properties underlies the existence of a ligand-linked structural change such that lactate binds oxygenated and deoxygenated Mb with different affinity, according to the following minimum reaction scheme (Scheme I) (13) ) observed upon addition of lactate (see Fig. 1) is due to a higher affinity of lactate for deoxyMb than for MbO 2 (i.e. K L o Ͼ K L d ), from which it stems that K O L Ͼ K O 0 (see Scheme I). The analysis of data reported in Fig. 1 according to Equation 1 is rendered more stringent by the observation that K L o can be obtained independently, since addition of lactate brings about a spectroscopic change of the absorption spectrum of sperm whale and horse heart MbO 2 , which is characterized by a decrease of the extinction coefficient at 418 nm (see Fig. 2A). Values of K L o turn out to be essentially the same (within the experimental error) for sperm whale and horse heart Mb, and the same can be said for K O 0 , that is the affinity for oxygen of the two Mb in the absence of lactate. Therefore, application of Equation 1 to data reported in Fig. 1, keeping fixed the values of parameters determined independently, allows a complete thermodynamic description of the effect of lactate on the O 2 binding properties of both Mbs (see Table I). It comes out that the different functional effect of lactate on sperm whale and horse heart Mbs (see Fig. 1) is totally related to a different affinity of lactate for the unliganded form of the two myoglobins.
As shown in Table I (Fig. 2B). Such behavior may indicate that either (i) lactate represents itself as a physical impairment for the dynamic approach of O 2 to the heme and/or (ii) a lactate- where Y is the molar fraction of the lactate-bound MbO 2 , and K L o is the equilibrium dissociation constant for lactate binding to MbO 2 . Values of K L o are reported in Table I. For further details, see text. B, Dependence of the pseudo-first order rate constant for O 2 binding to horse heart Mb (i.e. k obs ) on ligand concentration in the absence (E) and in the presence (q) of 0.2 M lactate, in 0.1 M MES, at pH 6.5 and 20°C. Continuous lines correspond to the nonlinear least-squares fitting of data calculated according to the following equation (Equation 3) where kЈ is the second order association rate constant for O 2 binding and k is the O 2 dissociation rate constant. Values of k (Ç) were fixed to those reported in Table I  crystal structure of different derivatives of sperm whale Mb has shown an anion (e.g. sulfate) binding site at hydrogen bonding distances from HisE7, ArgCD3, and ThrE10 residues (14). Soaking of sperm whale oxygenated Mb crystals in 0.1 M lactate at pH 6.5 did not result in significant changes of the protein electron density (at 2.0 Å resolution). Therefore, at this stage we have no direct evidence of lactate binding into a specific pocket, even though the functional effect indeed indicates (see Fig. 1) that the interaction does occur and that it affects the heme absorption features (see Fig. 2A). However, lack of lactate binding to the crystalline protein may be in keeping with the moderate affinity of lactate for MbO 2 (see Table I) as well as with the high ionic strength of the crystal mother liquor. In fact, crystals are grown in Ϸ4 M ammonium sulfate, i.e. an anion which at this concentration might efficiently compete with lactate for the HisE7-ArgCD3-ThrE10 binding site and remove it from the putative cleft in the distal side of the heme pocket. Sperm whale Mb shows at positions CD3 and E10 an arginyl and a threonyl residue, whereas in the case of horse heart Mb a lysine and a valine are present, respectively (14,15). Under the hypothesis of a possible interaction of lactate with the E7-CD3-E10 binding site, the different residues present at CD3 and E10 positions might be in keeping with the observed variation in the affinity of lactate for the deoxygenated form of the two Mbs (see Table I). Such a feature may suggest that in the unliganded form of Mb amino acid residues at positions CD3 and E10 are directly involved in the binding of lactate, and thus in the free energy of interaction with this exogenous ligand. On the other hand, the closely similar affinity of lactate for both hemoproteins in the oxygenated form (see Table I) seems to indicate that amino acid residues at positions CD3 and E10 are much less important for the interaction of lactate with liganded Mb. The effect of lactate on the functional properties of sperm whale and horse Mb has never been reported before and it has important physiological implications. As a matter of fact, during a prolonged physical effort, such as diving for sperm whale and running for horse, muscle cells are continuously consuming ATP, which cannot be fully reconstituted because of the decreased intracellular O 2 concentration (and the subsequent impairment of the oxidative phosphorylation). As a consequence, the glycolytic pathway progressively shifts from pyruvate to lactate production, ATP formation being further reduced. Hence, on the basis of the results reported here, lactate increase (e.g. from 1 mM to 10 mM (11,12)) may have useful consequences, since it may trigger a compensatory mechanism whereby the O 2 affinity of Mb is reduced (see Fig. 1) and the intracellular release of O 2 helps in keeping constant the ATP formation, even at O 2 pressures normally attained inside a cell (i.e. 2 to 5 mmHg oxygen).
As a whole, modulation of Mb function by a non-heme ligand envisages a mechanism which allows this molecule to be defined as an "allosteric monomeric protein," this observation being in line with recent reports on ligand-linked conformational changes occurring in Mb (5, 6).