Gino Amiconi

[23] Measurement of binding of gaseous and nongaseous ligands to hemoglobins by conventional spectrophotometric procedures

Publisher Summary This chapter presents an overview of the measurement of binding of gaseous and nongaseous ligands to hemoglobins by conventional spectrophotometric procedures. Ferrous hemoproteins bind a number of gaseous and nongaseous ligands specifically at the heme group. The reactions are fully reversible and may be described, at equilibrium, by standard thermodynamic relationships involving the application of the law of mass action. Equilibrium studies of the interaction of hemoglobin with heme ligands have been simplified by spectrophotometric procedures relating the changes in absorption spectrum to the degree of saturation of hemoglobin with ligands. The main assumption on which the spectrophotometric methods are based is that, within a tetramer partially saturated with a ligand, the spectral properties of the free and of the bound hemes correspond to those of the fully unliganded protein and of the fully liganded protein, respectively. Thus, in the quantitative evaluation of the ligand binding isotherm, linearity of the relationships between combination with ligand and spectral changes is assumed. The chapter describes gaseous ligands and nongaseous ligands.

The transition between ‘acid’ and ‘alkaline’ ferric heme proteins

We have studied the transition between the ‘acid’ and the ‘alkaline’ forms in several ferriheme proteins, which is generally attributed to the ionization of the water molecule coordinated with the heme iron in the sixth position. The different heme proteins studied (sperm whale and Aplysia myoglobins, Chironomus and human hemoglobins) have different dissociation constants, which arise mainly from heat effects. These differences are tentatively correlated to some differences in the general structure of the globin. Alkylation of sperm whale myoglobin or human hemoglobin, obtained by reaction with acetic or succinic anhydrides, leads to large changes in the pK′ of the transition. The profile of the curve relating the ΔpK′ to the percent alkylation suggests the possibility of dividing the different reactive groups of the protein into different classes on the basis of their effects on the ionization process. The substitution of hemes differing from protoheme in sperm whale myoglobin leads to significant changes in the equilibrium constant of the reaction, the pK′ ncreasing in the order proto-deutero-meso-myoglobins.

Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems

A novel method for glutathione-protein mixed disulphide (GSSP) determination, based on the use of protein sulphydryl groups as endogenous reductant and on the spectrophotometric determination of reduced glutathione, is described. The procedure is based on the observation that acid-precipitated proteins from different rat tissues rapidly release GSH from GSSP when brought to neutral pH. The basal GSSP content determined in rat liver, heart, lung, testis, spleen and brain corresponded to that reported in the literature and determined by more complex sample preparation or labor-intensive analytical procedures.

Artificial intermediates in the reaction of haemoglobin: Functional and conformational properties of the cyanmet intermediates☆

Artificial cyanmet intermediates of haemoglobin were obtained by mixing cyanmet chains with their oxygenated partners. At pH 7 and 20 °C, the oxygen affinity of the two intermediates is similar and lies between that of haemoglobin and the isolated chains; the value of n in the Hill equation is equal to 1 for (α + β+ CN) but may be slightly higher (~1.1 to 1.2) for (α+ N + β). The alkaline Bohr effect is very similar for the two intermediates, but the acid Bohr effect is lacking in (α + β+ CN). In the reaction with carbon monoxide, the intermediates are somewhat faster than haemoglobin but very much slower than the isolated chains. Conformational changes upon ligand binding to haemoglobin are implied by changes in reactivity of the β93 sulphydryl groups and by changes in the rotational strength at 233 nm. Addition of oxygen to (α+ CN + β) is followed by a change in the time-course of p-mercuribenzoate binding by the reactive sulphydryl groups, whereas oxygenation of the other intermediate does not modify the rate of mercuribenzoate binding. A change in the value of [R′] at 233 nm as a result of oxygen binding is observed, which is about the same in both cases (Δ [R′]233 ∼- 3%).

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Abstract Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10−3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum. The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li+, Ca2+ and Mg2+. The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10−2 m to 2 m. The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be −14·6 kcal/mol of O2.

Enzyme competitive inhibition. Graphical determination of Ki and presentation of data in comparative studies

In true competitive inhibition, substrate and inhibitor binding to the free enzyme are mutually exclusive and rates for substrate and inhibitor association and dissociation are never limiting in the process. Therefore, a competitive inhibitor acts only to increase the Michaelis constant for the substrate. Using the steady-state assumption, enzyme competitive inhibition may be described according to the well-known reaction scheme

Simultaneous static and dynamic light scattering approach to the characterization of the different fibrin gel structures occurring by changing chloride concentration

The structure of fibrin fibers has been investigated by simultaneous elastic and dynamic light scattering measurements. This approach allows the recovery of reliable structural parameters of the fibrin fibers while checking for the reliability of the necessarily ab initio assumptions on some nonexperimentally accessible parameters. The number of protofibrils per fiber section, N, related to the fiber diameter, and the gel mass fractal dimension Dm are obtained. Since the fiber size is largely controlled by ions in the gelling solution, a detailed characterization of the fiber structure formed by changing Cl− concentration is given. While N values decrease from 6000 to 1.5 protofibrils per fiber section, going from 0 to 300 mM in CCl−,Dm increases from 1 to 1.8. This means that the overall gel structure is affected by Cl−, being the fiber size decreased and branch points per unit volume increased.

Binding of the Recombinant Proteinase Inhibitor Eglin C from Leech Hzrudo Medzcznalzs to Human Leukocyte Elastase, Bovine α-Chymotrypsin and Subtilisin Carlsberg: Thermodynamic Study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)

Artificially induced unusual shape of erythrocytes: an atomic force microscopy study.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration.