Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of strong to ultratight protein interactions using differential scanning calorimetry

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.     

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes.

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The properties of the interaction between bovine pancreatic ribonuclease a and mononucleotides supports the existence of several binding sub-sites in the enzyme

The binding of 5'AMP, 5'GMP, 5'CMP, 3'CMP and Cl6RMP to RNAase A was studied by means of the gel filtration technique. It was found that only one molecule of 3'CMP binds strongly to the enzyme although a very unspecific binding is also present. The interaction of 5'AMP and 5'GMP with the enzyme shows one strong binding site and several weak binding sites, whereas two molecules of 5'CMP bind to RNAase A with equal strength. Cl6RMP shows an anomalous behaviour as both split peaks and troughs are found in the chromatogram. The Ka values for 3'CMP and the strong binding site of 5'AMP and 5'GMP are very similar whereas that for the two binding sites of 5'CMP is smaller (about 2.2 X 10(-4)M-1 and 0.5 X 10(-4)M-1, respectively at pH 5.5, I = 0.01 and 25 degrees C). The results are in general agreement with the known multiplicity of ligand-binding subsites in RNAase A.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium. Role of veratryl alcohol in polymer oxidation.

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase in strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 microM H2O2, relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1.s-1, compared to the second-order VA oxidation rate constant of 3.7 x 10(5) M-1.s-1. The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1.s-1, compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPIi are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

Cooperative interaction of histone H1 with DNA.

The cooperative binding of histone H1 with DNA was studied using a fluorescently labelled histone H1. The titration data were analysed in terms of the large ligand model. The stoichiometric number, n = 65 +/- 10 bases/H1, was independent of NaCl concentration (0.02 - 0.35 M). The nucleation and the cooperative binding constants, K' and K, and the cooperativity parameter q were sensitive to salt concentration; K = 3.6 +/- 0.8 X 10(7) M-1 and q = 1.1 +/- 0.4 X 10(3) at 0.2 M NaCl. The dependence of K' on NaCl concentration revealed that 6 Na+ ions were released from DNA upon complex formation. An extrapolation of K' to 1M NaCl yielded a small value, K' = 5 +/- 2 M-1. Thus the binding of H1 is essentially electrostatic, being compatible with its independence of temperature. A calculation of K' based on the counterion release reproduced the salt concentration dependence of K'. Therefore, the binding of H1 is of an electrostatic territorial type. Thus, H1 may move along the DNA chain to a certain extent, when both salt concentration and the degree of saturation are sufficiently low. The condition is so restricted that the sliding would not play an important role in vivo. It was concluded from the DNA concentration independent binding isotherm that H1 can cooperatively bind onto a single DNA molecule. A simple power law dependence of the cooperativity parameter q upon NaCl concentration was found; q oc[NaCl]h with h = 0.72, though the physical basis of this dependence remains unknown.     

Thermodynamics of the Ca2+ binding to bovine alpha-lactalbumin

Bovine alpha-lactalbumin contains one strong Ca2+-binding site. The free energy (delta G0), enthalpy (delta H0), and entropy (delta S0) of binding of Ca2+ to this site have been calculated from microcalorimetric experiments. The enthalpy of binding was dependent on the metal-free bovine alpha-lactalbumin concentration. At 0.8 mg ml-1, metal-free bovine alpha-lactalbumin delta H0 was -110 +/- 6 kJ mol-1. At this concentration the binding constant was estimated from a mathematical analysis of the titration curves to be greater than 10(7) M-1. This means that delta G0 is smaller than -40 kJ mol-1 and delta S0 is less negative than -235 J.K-1 mol-1. The binding of Ca2+ is therefore enthalpy-driven. From binding experiments as a function of temperature, a delta Cp value of -4.1 kJ.K-1 mol-1 was calculated. This value is dependent on the protein concentration. A tentative explanation for this large value is given.     

Valence and anion binding of bovine ribonuclease A between pH 6 and 8

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis-Tris propane over a pH range of 6.00-8.00. The results of data analysis using a Debye-Huckel-Henry model, compared with expectations based on pK(A) values, are consistent with the binding of two chlorides by RNase A. The decreased protein valence resulting from anion binding contributes 2-3 kJ/mol to protein stabilization. This work demonstrates the utility of first-principle valence determinations to detect protein solution properties that might otherwise remain undetected.     

The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy

The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis.     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Conformation of 3'CMP bound to RNase A using TrNOESY

The conditions for accurately determining distance constraints from TrNOESY data on a small ligand (3'CMP) bound to a small protein (RNase A, <14 kDa) are described. For small proteins, normal TrNOESY conditions of 10:1 ligand:protein or greater can lead to inaccurate structures for the ligand-bound conformation due to the contribution of the free ligand to the TrNOESY signals. By using two ligand:protein ratios (2:1 and 5:1), which give the same distance constraints, a conformation of 3'CMP bound to RNase A was determined (glycosidic torsion angle, chi=-166 degrees ; pseudorotational phase angle, 0 degrees < or = P < or =36 degrees ). Ligand-protein NOESY cross peaks were also observed and used to dock 3'CMP into the binding pocket of the apo-protein (7rsa). After energy minimization, the conformation of the 3'CMP:RNase A complex was similar to the X-ray structure (1 rpf) except that a C3'-endo conformation for the ribose ring (rather than C2'-exo conformation) was found in the TrNOESY structure.     

Enthalpic and entropic determinants for the specificity of alkylation of the histidine-12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide

The binding and alkylation rate constants for the reaction of four bromoacetamido pyrimidine nucleosides and bromoacetamide with bovine pancreatic ribonuclease A (RNase A) have been determined as a function of temperature. The four nucleoside derivatives react exclusively or preferentially with the NE2 atom of histidine-12 and include 2'-bromoacetamido-2'-deoxyuridine, 2'-bromoacetamido-2'deoxyxylofuranosyluracil, 3'-bromoacetamido-3'-deoxythymidine and 3'-bromoacetamido-3'-deoxyarabinofuranosyluracil. Transition-state parameters, delta H++ and delta S++, reveal that nucleosides with "up" OH groups experience relative rate enhancements which have been attributed to contacts between these groups and the enzyme in the transition state (1). Variations in alkylation rates are explained in terms of different degrees of entropic destabilization (2) of the nucleosides in the enzyme.affinity label complex.     

RNase A - tRNA binding alters protein conformation.

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5' bonds in RNA on the 3' side of pyrimidine to form cyclic 2',5'-phosphates. Even though extensive structural information is available on RNase A complexes with mononucleotides and oligonucleotides, the interaction of RNase A with tRNA has not been fully investigated. We report the complexation of tRNA with RNase A in aqueous solution under physiological conditions, using a constant RNA concentration and various amounts of RNase A. Fourier transform infrared, UV-visible, and circular dichroism spectroscopic methods were used to determine the RNase binding mode, binding constant, sequence preference, and biopolymer secondary structural changes in the RNase-tRNA complexes. Spectroscopic results showed 2 major binding sites for RNase A on tRNA, with an overall binding constant of K = 4.0 x 105 (mol/L)-1. The 2 binding sites were located at the G-C base pairs and the backbone PO2 group. Protein-RNA interaction alters RNase secondary structure, with a major reduction in alpha helix and beta sheets and an increase in the turn and random coil structures, while tRNA remains in the A conformation upon protein interaction. No tRNA digestion was observed upon RNase A complexation.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: 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 (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency.