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Biomedical subjects

M C Marden

Publications and source records attributed to M C Marden.

At least 19 recordsLinked to original sources
PubMed1995

Loss of allosteric behaviour in recombinant hemoglobin alpha 2 beta 2(92)(F8) His-->Ala: restoration upon addition of strong effectors.

In the stereochemical model proposed by Perutz [1], the Fe-His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO)4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy-like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the alpha 2 beta 2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R-state. However, a significant amount of deoxy (T-state) kinetics are observed after addition of external effectors such as IHP. The iron-histidine bond is apparently crucial for the heme-heme interaction, but the allosteric equilibrium may still be influenced by external constraints.

Alanine↗
PubMed1995

Heme as an optical probe for studying the interactions between calmodulin and the Ca(2+)-ATPase of the human erythrocyte membrane.

The heme group was used as an optical probe to study the interactions between calmodulin and its targets: the peptide melittin and the enzyme Ca(2+)-ATPase. As already reported, melittin when present in Tris buffer binds hemin-CN which quenches the tryptophan fluorescence. Addition of calmodulin restores the fluorescence significantly accompanied by a blue shift. We show here that the recovery of fluorescence is very slow and takes about 120 min to become constant. In a hydrophobic buffer, the fluorescence spectrum of melittin is already shifted with a peak at 335 nm and intensity almost 2-fold relative to a similar concentration of melittin in Tris buffer. The quenching of tryptophan fluorescence is lesser in this buffer and further addition of calmodulin fails to restore the fluorescence. This indicates the absence of binding of calmodulin to melittin in hydrophobic conditions. Under similar conditions of hydrophobicity, hemin-CN quenches about 35% of the tryptophan fluorescence of the Ca(2+)-ATPase. The subsequent addition of calmodulin restores about half of the quenched fluorescence. The interaction of calmodulin with the Ca(2+)-ATPase even under hydrophobic conditions suggests its high specificity for the enzyme which may be expected for a physiological target.

Calcium-Transporting ATPases↗
PubMed1995

Intrinsic activity at the molecular level: E. J. Ariëns' concept visualized.

The concept of using affinity and intrinsic activity to analyze drug interactions with receptors has had a long history in pharmacological studies. In the simplest case, the biological response will be proportional to the amount of drug bound, i.e. its affinity. However, the biological response is also mediated by the ability of a drug when bound to exert its maximum effectiveness. This effectiveness is termed the intrinsic activity. Physicochemical processes have been thought to be at the basis of intrinsic activity. Detailed oxygen and solution binding experiments combined with X-ray crystallographic studies on allosteric effectors to hemoglobin demonstrate that these potential drug agents bind at the same site in hemoglobin with similar binding constants yet shift the allosteric equilibrium and the oxygen affinity of the T-structure by different degrees. Therefore some of the effectors with similar binding affinities for the same site exhibit varying degrees of affectiveness, i.e. they possess different intrinsic activities. The intrinsic activity of the effector is defined as the ratio of the oxygen affinity constant to the T-state with drug/oxygen affinity constant to the T-state without drug (KT+drug)/(KT control). The source of the intrinsic activity appears to be the ability of the effectors to interact with key residues such as Lys99 alpha at the binding site. These results suggest a general molecular mechanism for allosteric effector modulation of hemoglobin function that might be of use in other allosteric enzyme systems.

Allosteric Regulation↗
PubMed1995

Correlation of carbon monoxide association rates and the position of absorption band III in hemoproteins.

We have examined the absorbance of a charge-transfer transition near 760 nm, known as band III, in several hemoproteins and heme complexes. The band III position correlates with the rate of carbon monoxide binding to the heme. A band III present at 760 nm indicates an unfavorable geometry of the heme for carbon monoxide binding; a red-shift of the band III to 765 nm indicates a less-constrained geometry of the heme as evidenced by higher carbon monoxide association rates. The band III position correlates well with the Raman frequency of the Fe-His(F8) bond as suggested previously for normal hemoglobin A [Sassaroli, M. & Rousseau, D. L. (1987) Biochemistry 26, 3092-3098]. Aplysia myoglobin and the chimeric heme protein kinase FixL from Bradyrhizobium japonicum, hemoproteins with an apolar residue in place of the highly conserved polar histidine E7, do not fit the relationship between the band III position and the rate of binding of carbon monoxide to the heme. With these few exceptions, the measurement of band III appears to be a practical means to probe the stretch frequency of the Fe-His(F8) bond.

Carbon Monoxide↗
PubMed1994

Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation.

FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.

Bacterial Proteins↗
PubMed1994

Formation of two hydrogen bonds from the globin to the heme-linked oxygen molecule in Ascaris hemoglobin.

We have tried to find out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1 mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J., Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like Kloek et al., we found its oxygen affinity to be as high as that of native Ascaris hemoglobin. We thought that this high affinity might be due to the heme-bound oxygen molecule being stabilized by two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in place of the leucine generally found there in vertebrate myoglobins and hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr., & Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these replacements lowered the oxygen affinity about 100-fold, to the same level of that of human alpha-globin chains. These results are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.

Allosteric Regulation↗
PubMed1994

Binding of heme-CO to bovine and porcine beta-lactoglobulins.

Two molecules of heme-CO bind to bovine or porcine beta-lactoglobulin (BLG) with an average affinity of 0.5 microM. The affinity increases with pH, with a transition near pH 7.5, indicating a conformational change in the protein. Heme-CO does not bind to the predominantly alpha-helix conformation of BLG, which occurs in solutions with more than 40% ethanol or methanol. Fluorescence energy transfer measurements have been made for the complex of BLG with retinol and/or heme-CO. Two species of BLG were used. While bovine BLG possesses two tryptophans (at positions 19 and 61) which are quenched by about a factor of 2 by either retinol or heme-CO, the porcine species has only one tryptophan (at position 19) whose fluorescence is decreased by a factor of 15 when both hemes are bound, indicating that at least one of the heme-binding sites is near (< 20 A) to this tryptophan. The fluorescence of retinol (complexed to BLG) is also quenched by the addition of heme-CO, indicating that BLG can bind both molecules simultaneously; a separation of 25 A between retinol and heme was calculated. The results suggest at least two hydrophobic pockets for this protein.

Animals↗
PubMed1993

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

Allosteric Site↗
PubMed1993

Interaction of heme with amphiphilic peptides: use of hemin-CN to probe the interaction of calmodulin with its target peptides.

UNLABELLED: The interaction of heme with several amphiphilic peptides has been studied by absorption and fluorescence spectroscopy. The binding can be followed by the changes in the absorption spectrum of the heme group or by the decrease in the peptide tryptophan fluorescence due to energy transfer to the heme. Despite their small size, ranging from 26 residues for melittin to 14 for mastoporan, a high affinity for heme-CO and hemin-CN (Kd < 100 nM) may be observed. Spectral shifts in the absorption peaks and appreciable geminate recombination after photodissociation of CO from the complex peptide-heme-CO suggest the formation of a heme pocket, as for the natural heme proteins. APPLICATION: hemin-CN can be used as a probe for the interaction of calmodulin with these target peptides. Amphiphilic peptides such as melittin bind to calmodulin with a high (nM) affinity. While both the peptide and calcium-bound calmodulin bind heme-CO, only the peptide binds hemin-CN. These interactions permit studies of the competition between hemin-CN and calmodulin for binding to the peptide: while hemin-CN quenches the melittin tryptophan fluorescence, addition of calmodulin to the [melittin*hemin-CN] complex displaces the hemin-CN and the melittin tryptophan fluorescence is recovered.

Amino Acid Sequence↗
PubMed1993

Heme binding to calmodulin, troponin C, and parvalbumin, as a probe of calcium-dependent conformational changes.

Heme-CO binds to the active (calcium-bound) form of calmodulin (CaM), but not to the inactive form. Despite a similarity in structure of another calcium-binding protein, skeletal muscle troponin C, both the affinity and the spectral red-shift of the absorption of the heme group are greatly decreased for troponin C relative to calmodulin. Parvalbumin, another calcium-binding protein, shows a twofold greater affinity for heme-CO relative to CaM. Unlike calmodulin and troponin C, the affinity of parvalbumin for heme-CO is even greater in the absence of calcium. The affinity of the tryptic and thrombic fragments of CaM for heme-CO are decreased relative to the entire calmodulin. The binding of heme-CO is specific as demonstrated by the discrimination of the calmodulin, troponin C, and parvalbumin pockets. The interaction of heme-CO with active (calcium-bound) CaM is rapid (ms) as determined by stopped flow measurements. No difference in kinetics was observed for mixing inactive (calcium free) CaM with a solution of [heme-CO plus calcium], indicating that the calcium-binding step and subsequent change in protein conformation are rapid.

Animals↗
PubMed1993

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains. Samples of symmetric hybrids (a2NOI32Co or a2Co922NO) showed a lower (R-like) bimolecular yield and less slow phase for the CO bimolecular recombination, relative to the asymmetric hybrid or the triply NO species. The slower (T state) bimolecular rate of 7 x 104 M-1 s-1 was observed for CO rebinding to a chain.While oxygen equilibrium studies with 'HPHb(NO)3 were hampered by a high oxidation rate, it was possible to perform experiments with samples equilibrated with a mixed CO/oxygen atmosphere. Photodissociation of CO allows a temporary exposure of the binding sites to oxygen. The results confirm that IHPHb(NO)3 has a low oxygen affinity.

Allosteric Regulation↗
PubMed1993

Hb Val de Marne [alpha 133(H16)Ser-->Arg]: a new hemoglobin variant with moderate increase in oxygen affinity.

Hb Val de Marne [alpha 133(H16)Ser-->Arg] was found in a French family during a neonatal hemoglobinopathy screening program. The abnormal hemoglobin was found, within a few months interval, in two newborn children who were first cousins. In the children as well as in the parents carrying this hemoglobin variant, the red cell parameters were normal. Hb Val de Marne, isolated by isoelectrofocusing, displayed a slightly higher auto-oxidation rate than Hb A. Its oxygen affinity was increased 1.7-fold in comparison to that of Hb A. The heterotropic effects were normal.

Adult↗
PubMed1991

Allosteric transition in triply met-haemoglobin.

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.

Allosteric Regulation↗
PubMed1991

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.

Allosteric Regulation↗
PubMed1990

Beta-lactoglobulin binds retinol and protoporphyrin IX at two different binding sites.

Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that beta-lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand-to-protein stoichiometric ratio. The apparent dissociation constants of hemin-beta-lactoglobulin and protoporphyrin IX-beta-lactoglobulin complexes are 2.5 x 10(-7) M and 4 x 10(-7) M, respectively. The addition of beta-lactoglobulin (final concentration = 10 microM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between beta-lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Förster theory, a retinolprotoporphyrin IX distance of 25 A was calculated. These results indicate that retinol and protoporphyrin IX are bound to the beta-lactoglobulin monomer at two different sites.

Animals↗