Calorimetric and optical characterization of sickle cell hemoglobin gelation.

We have used two techniques to characterize the gelation of deoxyhemoglobin S, a high sensitivity heat-flow calorimeter to measure the heat of gelation and a simple light-transmission method to measure the optical birefringence resulting from the alignment of deoxyhemoglobin S fibers in the gel. A theory for the interpretation of the birefringence measurements is presented. We combine the results of the calorimetric and optical measurements with those of sedimentation experiments to obtain enthalpy changes for gelation. The enthalpy change obtained from scanning and isothermal calorimetric measurements (0.25 m-potassium phosphate, 0.05 m-sodium dithionite, pH 6.9) varies from 4000 to 2200 cal mol⁻¹ hemoglobin between 16 and 25 °C. There is a large apparent heat capacity change of −130 to −190 cal deg.⁻¹ mol⁻¹. The apparent enthalpy change estimated from solubility measurements and birefringence melting experiments is 2200 ± 500 cal mol⁻¹ in qualitative agreement with the calorimetric results. Analysis of the time dependence of the calorimetric and optical progress curves at 20 °C leads to a rough estimate of 1800 to 4000 and −800 to 1500 cal mol⁻¹ hemoglobin for the enthalpies of polymerization and alignment of fibers, respectively. The small magnitude of the observed enthalpy change is in accord with the view that no large conformational change takes place in the deoxyhemoglobin S molecule upon gelation.     

Thermodynamics of gelation of sickle cell deoxyhemoglobin.

This paper describes the thermodynamic behavior of gels of deoxyhemoglobin S. The solubility of the protein with respect to assembled hemoglobin fibers has been measured using a sedimentation technique. The solubility in 0.15 m-potassium phosphate buffer (pH 7.15) is found to decrease with increasing temperature, attain a minimum value of 0.16 g cm^?3 at 37 °C, and then increase at higher temperatures. The amount of polymer present at various hemoglobin concentrations and temperatures is presented as part of a phase diagram that may be useful for the calibration of other measurement techniques. The effects of varying pH and urea concentration upon the solubility have also been studied.The heat absorption accompanying gelation has been measured by scanning calorimetry. Using sedimentation data on the amount of polymer formed, molar enthalpy changes are obtained. There is a large negative heat capacity change of ? 197 cal deg. mol^?1 and ΔH = 0 near 37 °C. Calorimetric molar enthalpy changes are found to agree with those calculated from the temperature dependence of the solubility by the van't Hoff equation.Our previous two-phase, two-component thermodynamic model of gelation is extended to include the effects of solution non-ideality. A large contribution to the activity of the hemoglobin in the solution phase results from the geometric effect of excluded volume. Incorporating solution phase non-ideality permits the calculation of standard state thermodynamic quantities for the gelation process at 37 °C: ΔG^O ? ?3 k cal mol^?1, ΔH^O ~ 0, ΔS^O ~ 10 cal deg.^?1 mol^?1. The excluded volume effect is also capable of explaining observations of the minimum gelling concentrations of hemoglobin mixtures containing deoxyhemoglobin S without requiring copolymerization of the non-S hemoglobin.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

Temperature-jump kinetics of the dC-G-T-G-A-A-T-T-C-G-C-G double helix containing a G . T base pair and the dC-G-C-A-G-A-A-T-T-C-G-C-G double helix containing an extra adenine

The kinetics of helix formation were investigated using the temperature-jump technique for the following two molecules: dC-G-T-G-A-A-T-T-C-G-C-G, which forms a double helix containing a G·T base pair(the G·T 12-mer), and dC-G-C-A-G-A-A-T-T-C-G-C-G, which forms a double helix containing an extra adenine (the 13-mer). When data were analyzed in an all-or-none model, the activation energy for the helix association process was 22 ± 4 kcal/mol for the G·T 12-mer and 16 ± 7 kcal/mol for the 13-mer. The activation energy for the helix-dissociation process was 68 ± 2 kcal/mol for the G·T 12-mer and 74 ± 3 kcal/mol for the 13-mer. Rate constants for recombination were near 10⁵s^?1M^?1 in the temperature range from 32 to 47°C; for the dissociation process, the rate constants varied from 1s^?1 near 32°C to 130s^?1 near 47°C. Possible effects of hairpin loops and fraying ends on the above data are discussed.     

The standard Gibbs free energy change of hydrolysis of alpha-D-ribose 1-phosphate

The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H₂O ág adenine + ribose (ΔG^0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + P_i (ΔG^0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.     

Effects of pH, 2,3-diphosphoglycerate and salts on gelation of sickle cell deoxyhemoglobin

Gelation of fully deoxygenated sickle cell hemoglobin was assayed by (1) determination of the temperature at which viscosity increased sharply and (2) a high-speed sedimentation equilibrium method in which three zones are seen. These are a pre-gelation zone, a narrow transition zone exhibiting aggregation, followed by a phase change and a zone of gelation. Only the first zone is seen with deoxyhemoglobin A and CO hemoglobins A and S up to about 0·35 g protein/ml. Minimal gelling temperatures by the viscosity method and, by ultracentrifugation, minimal gelling concentrations determined at the onset of aggregation and at the phase change showed: (a) lowering the pH toward 6·7 favors gelation; (b) deoxyhemoglobin S gels more readily in 6 mm-2,3-diphosphoglycerate than in its total absence; (c) 1 m-NaCl and l m-KCl inhibit gelation. The known favoring of gelation by warming is confirmed by the equilibrium method and is about 2% change in minimal gelling concentration per degree.The effects of pH and high ionic strengths are consistent with contributions of specific polar interactions to gel structure. The effect of 2,3-diphosphoglycerate probably depends on known structural changes which this cofactor induces rather than on alteration of the allosteric quaternary structure equilibrium.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Packaging of DNA in bacteriophage heads: some considerations on energetics

We have made quantitative estimates of some of the energetic factors to be considered in packaging of double-stranded DNA in virus particles. Numerical calculations were made using parameters appropriate for T4 bacteriophage. The unfavorable factors, and the Gibbs free energies per mole virus at 20°C associated with them, are bending, 1.5 × 10³ kcal/mol; conformational restriction upon condensation, 5.1 × 10² kcal/mol; polyelectrolyte repulsion, 2.1 × 10⁵kcal/mol; and melting or kinking, 6.9 × 10³ kcal/mol. These must be counterbalanced in the assembled phage by noncovalent bonding interactions between protein subunits in the phage-head shell; by interactions between the DNA and polyvalent cations, especially putrescine and spermidine; nad perhaps by repulsive excluded volume and electrostatic interaction between the DNA and acidic polypeptides. Indeed, a rough estimate of the standard free energey of interaction between T4 DNA and the putrescine and spermidine contained in the head is --2.1 × 10⁵ kcal/mol. In the absence of the other two sources of stabilization, each head protein subunit must contribute about 210 kcal/mol of binding energy.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.     

Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins.

We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 10⁷ relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 10⁴. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α₂γ₂) and hybrid α₂γβ^S molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α₂β^Aβ^S molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the $\text{S}\text{F}$ mixtures can be explained by assuming that only α₂β₂^S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α₂β^Aβ^S hybrid molecule and the absence of any significant copolymerization of the α₂γβ^S hybrid.     

Microcalorimetric method to determine competitive binding action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan · human serum albumin complex

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated.The association constants and the enthalpy variations are equal to 14 000 ± 2000 M^?1 and ?6.6 ± 0.2 kcal/mol for human serum albumin · tryptophan complex and 13 000 ± 1000 M^?1 and ?10.0 ± 0.2 kcal/mol for human serum albumin · chlorazepate complex. In all cases the stoichiometry is equal to one.The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 ± 200 M^?1 and ?2.6 ± 0.3 kcal/mol.     

The interaction of albumin and concanavalin A with normal and sickle human erythrocytes.

The interaction of human albumin and concanavalin A with normal and sickle human red blood cells previously washed in phosphate buffer at pH = 7.4 was studied by titration calorimetry. The amount of albumin bound to normal cells was (6.8 ± 2.2) × 10⁵ molecules/cell. An equilibrium constant of 5 × 10¹⁰ and an enthalpy change of ?(280 ± 90) kcal/mol albumin was determined for albumin interaction with normal cells. The amount of albumin bound to sickle cells was (12.4 ± 1.0) × 10⁵ molecules/cell and the enthalpy change for albumin interaction with sickle cells was ?(390 ± 140) kcal/mol. Normal cells bound (5.7 ± 2.4) × 10⁵ concanavalin A molecules/cell with an enthalpy change of ?(840 ± 200) kcal/mol concanavalin. All experiments were conducted at 25°C.     

Tissue Oxidative Capacity,Fuel Stores and Skeletal Muscle Fatty Acid Composition In Obesity-Prone and Obesity-Resistant Rats

The purpose of the present study was to compare tissue oxidative capacity, skeletal muscle fatty acid composition, and tissue fuel stores in low-fat fed (LFD, 12% of energy from corn oil) male Wistar rats, and in high-fat fed (45% of energy from corn oil) obesity-prone (OP) and obesity-resistant (OR) male Wistar rats. Designation of OP and OR rats was based on body weight gain (upper tertile for OP; lower tertile for OR) after 5 weeks on the high-fat diet. Body weight gain over the 5-week dietary period was 91 ± 9 g in LFD, 98 ± 4 g in OR, and 158 ± 5 g in OP (p<0. 05 vs. LFD and OR). Energy intake over the 5-week dietary period was 3099 ± 101 kcal in LFD, 3185 ± 51 kcal in OR, and 3728 ± 45 kcal in OP (p<0. 05 vs. LFD and OR). Maximal citrate synthase activity (μ. mol^?1min^?1) in the gastrocnemius muscle was not significantly different among groups: 12. 1 ± 2. 4 in LFD, 11. 4 ± 1. 9 in OR and 133 ± 2. 5 in OP rats. Similarly, citrate synthase activity in the heart, 59. 3 ± 7. 2, and liver, 6. 6 ± 0. 4, was also not significantly different among groups. Fatty acid composition of the gastrocnemius muscle was not significantly different among groups. Fasting glycogen levels in the liver, gastrocnemius muscle, and heart were 6. 4 ± 3. 7, 13. 2 ± 2. 3 and 6. 8 ± 1. 9 μmol/g in LFD, 21. 2 ± 5. 1 (p<0. 05 vs. LFD and OP), 10. 4 ± 1. 8 and 5. 9 ± 1. 1 mUmol/g in OR, and 36. 3 ± 4. 8 (p<0. 05 vs. LFD and OR), 10. 2 ± 23 and 53 ± 2. 1 μmol/g in OP rats, respectively. Triglyceride levels were similar among groups in plasma, heart and gastrocnemius muscle, but were significantly (p<0. 05) higher in the liver of OP (15. 5 ± 1. 9 (μmol/g) compared to OR (9. 1 ± 1. 1 μmol/g) and LFD (8. 1 ± 1. 4 μmol/g) rats. These data suggest that susceptibility to dietary obesity, in this rodent model, cannot be explained by differences in tissue oxidative capacity or muscle fatty acid composition.     

Gelation of sickle cell hemoglobin IV. Phase transitions in hemoglobin S gels: Separate measures of aggregation and solution-gel equilibrium

Two assays of equilibrium properties in the gelation of deoxyhemoglobin S were carried out by analytical ultracentrifugation on the same sample: C_sat, the monomer concentration in equilibrium with the fully formed gel, was obtained as the supernatant concentration after sedimentation of a preformed gel. The presence of a plateau region during sedimentation of the supernatant and the rate of sedimentation of the boundary from which C_sat was measured indicate that centrifugation did not alter the pre-existing equilibrium and that the supernatant consisted of monomers. The centrifugation was then continued to equilibrium to obtain a distribution showing a sharp increase in molecular weight at C_agg, the monomer concentration at which a small amount of polymerization to large aggregates begins.The primary result is that C_sat > C_agg under all conditions. The different values of the two parameters indicate that they reflect two separate transitions and that the overall monomer to gel process has a limited co-operativity. Within the limits of the method C_sat is independent of total hemoglobin concentration. The two transitions divide the overall range of total hemoglobin concentration into an essentially monomeric region at concentrations below C_agg, a region in which isotropically oriented polymers exist, occurring when monomer concentration lies between C_agg and C_sat, and a two-phase region of conjugate isotropic and anisotropic phases when monomer concentration equals C_sat. These regions correspond to zones in the ultracentrifuge equilibrium distribution. In this scheme C_agg depends only on the interaction energy of polymerization. C_sat depends on entropic factors which induce tactoid formation as well. C_sat, while a monomer concentration, reflects a saturation not of monomers in relation to a polymeric phase, but of polymers in the isotropic phase in relation to the anisotropic or tactoidal polymerized phase. As such, C_sat represents a supersaturated state of isolated monomers.The ratio $\text{C}{}_{\mathrm{<mtext>sat</mtext>}}\text{C}{}_{\mathrm{<mtext>agg</mtext>}}\text{= 1.23}$ in stripped hemoglobin³ and equilibrium distributions in the zone of isotropically oriented polymers were both used to obtain an order of magnitude estimate of polymer size, found to be much smaller than that of hemoglobin S fibers. This further confirms that gelation does not consist of a single transition and phase change with near infinite co-operativity of polymerization.C_sat as well as C_agg are lowered by 2,3,diphosphoglycerate and inositol hexa-phosphate. Decreasing pH near 7 also favors gelation; in stripped hemoglobin a pH optimum for gelation occurs near pH 6.8. The apparent van't Hoff ΔH for stripped hemoglobin is about 3 kcal/mol for C_sat and 2 kcal/mol for C_agg.     

Demonstration of lag phase in the sol-gel transformation of deoxygenated S hemoglobin without temperature alteration.

1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

Identification and Inhibitory Mechanism of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Bovine Hemoglobin

Angiotensin I-converting enzyme (ACE, EC.3.4.15.1) inhibitory peptide is an efficacious therapy for hypertension. In this study, four dipeptides, TY, FD, FL and FG, were identified from the desalted fraction of bovine hemoglobin hydrolysate, obtained by in vitro simulated gastrointestinal digestion, via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The IC₅₀ value of TY and FL are 96.43?±?6.17 and 290.66?±?57.92 μM, respectively. The result of molecular docking indicated that TY occupied the ACE subsite S1 and S1′ with a lowest estimated binding energy of ?9.96 Kcal/mol, while FL occupied the subsite S5 with a lowest estimated binding energy of ?9.37 Kcal/mol. The subsite S1′ and S2′ are closer to the ACE active center (Zn²⁺) than S5, and the lowest estimated binding energy of TY is lower than that of FL. This work provided new ACE-inhibitory peptides derived from bovine hemoglobin hydrolysate and explained their inhibitory mechanism.     

Correlations of blood selenium with hematological parameters in West German Adults

The serum selenium and the whole blood selenium of 72 healthy persons (47 women, 25 men) was determined. There exist sex specific differences of the whole blood selenium between men (98±19 μg Se/L) and women (89±17 μg Se/L). The serum selenium did not show sex specific differences, but sex specific differences are found if the total amount of extracellular selenium is calculated by correction of the serum selenium with the hematocrit. Women have more extracellular selenium/L whole blood (40±8 μg Se) than men (36±7 μg Se). Men have more intraerythrocyte selenium (cellular selenium=67±14 μg Se) in one L whole blood than women (52±17 μg Se). There exist also sex specific differences if the cellular selenium is calculated/g hemoglobin (men .44 μg Se/g Hb, women .37 μg Se/Hb) or per erythrocyte (men 136.1×10^?19 g Se/Ery, women 113.9×10^?19 g Se/Ery). In the cellular compartment of one L whole blood on the average 1.56 times more selenium is present than in the extracellular compartment. Most of the intraerythrocyte selenium is hemoglobin bound (84%) and utmost 16% glutathione peroxidase associated. An erythrocyte contains about 3500 mol glutathione peroxidase, or, for every 80000 mol hemoglobin one mol glutathione peroxidase. A standard man needs about 2.5 μg selenium/d for the synthesis of the hemoglobin and the erythrocyte. The hematological parameters hemoglobin and the erythrocyte number are correlated with the cellular selenium and the ratio cellular selenium/extracellular selenium. Positive significant correlations are found that are best if a parabolic model is used to interpret the shape of the curves. From the shape of the best correlation lines it can be concluded that selenium may be beneficial for hemoglobin synthesis and erythropoesis. The extracellular selenium may have influence on the volume of the erythrocyte by protecting the outer erythrocyte membrane from lipid peroxidation. A method is reported based on the carbon furnace atomic absorption spectroscopy, which is able to determine without wet digestion selenium in whole blood.     

Molecular Modeling of Cloned <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Keratinase and Its Insinuation in Psoriasis Treatment Using Docking Studies

Present study demonstrated the expression of cloned Bacillus subtilis RSE163 keratinase gene and in silico binding affinities of deduced protein with psoriasis topical drugs for systemic absorption and permeation through skin. The ker gene expressed in E. coli showed significantly higher keratinase activity 450 ± 10.43 U representing 1342 bp nucleotides encoding 447 amino acids with molecular weight of 46 kDa. The modeled structure was validated using ramachandran’s plot showing 305 residues (84.3%) in most favoured region. Docking studies using extra precision (XP) method of Glide showed optimum binding affinities with the drugs Acitretin (? 39.62 kcal/mol), Clobetasol propionate (? 37.90 kcal/mol), Fluticasone (? 38.53 kcal/mol), Desonide (? 32.23 kcal/mol), Anthralin (? 38.04 kcal/mol), Calcipotreine (? 21.55 kcal/mol) and Mometasone (? 28.40 kcal/mol) in comparison to other psoriasis drugs. The results can further be correlated with in vitro enzymatic experiments using keratinase as an effective drug mediator through skin to serve the unmet need of industries.