Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis

The specificity ratios kc/Km = k for subtilisin A catalyzed hydrolysis of five aryl esters of N-(methoxycarbonyl)-L-Phe (McPhe) were determined at pH 7.03 and its pD equivalent. The ratios are independent of the electronic properties of the leaving group substituent. Kinetic solvent isotope effects, Dk, increase from about 0.9 to 1.3 as leaving group ability decreases from p-nitrophenolate to p-methoxyphenolate. The k of N-(methoxycarbonyl)-L-phenylalanine p-nitrophenyl ester (NPE) with native enzyme exhibits a strong temperature dependence; delta H* = 87 +/- 3 kJ mol-1 and delta S* = 148 +/- 14 J K-1 mol-1 at 25 degrees C (H2O). The Dk with this substrate is 1.36 at 13.6 degrees C, declines to 0.89 at 25 degrees C, and then increases to 1.04 at 39.4 degrees C. Above neutral pH(D), with McPhe NPE as substrate, the dependence of k is for the dissociated form of a single base of pKapp = 7.38 +/- 0.03 in H2O and 7.67 +/- 0.03 in D2O. The pKapp values are apparently those of the uncomplexed native protein. By contrast, k of 3-phenylpropanoic acid (Prop) p-nitrophenyl ester exhibits a weaker temperature dependence; delta H* = 20 kJ mol-1 and delta S* = -90 J K-1 mol-1 (H2O) at 25 degrees C. The Dk are larger than those for McPhe NPE, decreasing from 1.99 at 20.5 degrees C to 1.74 at 46.1 degrees C. These results, combined with those of previous studies, are consistent with limitation of k by at least two processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation and aggregation of lactic dehydrogenase by high hydrostatic pressure

As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and --SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Lüdemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90--98; (1978) Eur. J. Biochem. 86, 219--224]. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation (less than or equal to 10 min) at pH 6.0--8.5 and pressures of 0.3--1.0 kbar causes dissociation which is reversed at normal pressure. At 5 degrees C the activation volume is found to be delta V not equal to = -62 +/- 3cm3 . mol-1. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5 degress C is delta V not equal to = -97 +/- 3cm3 . mol-1. The results may be described by a reaction sequence comprisign pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive high-molecular-weight particles.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.     

Covalent binding of a carcinogen as a probe for the dynamics of deoxyribonucleic acid

In order to determine the kinetic parameters of the binding to DNA of two closely related ultimate carcinogens, 2-(N-acetoxy-N-acetylamino)fluorene (N-Aco-AAF) and 2-(N-hydroxyamino)fluorene (N-OH-AF), three kinds of experiments were performed: measurement of the final amount of adduct (N-Aco-AAF and N-OH-AF), determination of the initial rate, and study of the reaction with deoxyguanosine (N-Aco-AAF only) at temperatures between 4 and 50 degrees C. The kinetic treatment of the chemical equations relies on two main assumptions: (i) binding of carcinogen to the C8 of guanine (G) could occur either with the classical B conformation or with a transient conformational state of the sugar--phosphate chain at the level of the guanine and denoted by G*; (ii) the equilibrium between G and G* is fast as compared to the chemical rate of carcinogen binding. These two assumptions have been verified by comparing experimental and calculated values of some of the data. From experimental data it is possible then to determine the characteristic independent parameters of the reaction: the constant K of the G in equilibrium G* and the enthalpy change delta H of the process, the rate constant k3 of the binding to the C8 of G, and the rate constant k1 of hydrolysis of the carcinogen with their corresponding activation enthalpies E3 and E1. Some essential results obtained are as follows: (a) The amount of G* that represents about 10% of the G at room temperature increases with temperature and is higher in denatured than in native DNA. (b) The values of delta H (approximately 9 kcal mol-1) and delta S (approximately 27 cal K-1 mol-1) of the G in equilibrium G* equilibrium are close to those associated with single base pair opening [Wartell, R.M., & Benight, A.S. (1982) Biopolymers 21, 2069]. (c) N-Aco-AAF reacts only with the G* conformation while N-OH-AF binds preferentially to the "classical" G (B conformation). Therefore, the electrophilic carcinogens behave as probes of the dynamic state of the DNA, but the rate of the G in equilibrium G* exchange is fast as compared to the binding rate of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

Biochemical characterization of positive cooperativity in the binding of 1 alpha, 25-dihydroxyvitamin D3 to its chick intestinal chromatin receptor

We have characterized a positive cooperativity mechanism in the binding of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to its chick duodenum chromatin receptor. The Hill plot which can take account of the possibility of cooperativity resulted in a much better fitting of the experimental data than the Scatchard model (r = +0.998 versus r = -0.94). Concentrating the chromatin receptor preparation from 10 to 40% resulted in an increase of the Hill coefficient (nH) from 1.09 +/- 0.08 to 1.46 +/- 0.08 (S.D.). Increasing the temperature of incubation from 1 degree C to 40 degrees C resulted in a decrease of nH from 1.46 +/- 0.08 to 1.10 +/- 0.02 (S.D.). The calculation of the thermodynamics of the interaction of 1,25-(OH)2D3 with the second binding site of the receptor (from a Van't Hoff plot) showed that this process occurred spontaneously (delta G0 = -11.6 kcal X mol-1 at 1 degree C), was entropy-driven (delta S0 = +26 cal degree-1 mol-1), and was energy-requiring (delta H0 = -4.37 kcal X mol-1). The temperature controlled reversibility of the cooperativity demonstrates that this phenomenon is not an artifact. Finally, in a study of the rate of dissociation of [3H]1,25-(OH)2D3 from the duodenal receptor preparation, we have found two slopes (k-1 = 32 X 10(-3) min-1; k-2 = 3.2 X 10(-3) min-1); this suggests the existence of two species of receptor. These receptor species could result possibly from either a monomer-dimer system or from a conformational change of a monomer via site-site interactions. In conclusion, the positive cooperativity in the binding of 1,25-(OH)2D3 to the two binding sites of its intestinal receptor is an entropy-driven process and requires energy, is reversible with temperature, and has been shown to take place in concentrated chromatin aggregates.     

Pressure effects on substrate activation phenomena in the alpha-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core.

The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

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.     

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.     

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Thermal inactivation and stabilization of lysozyme substrate-- Micrococcus lysodeicticus cells

Heat inactivation of the acetonic powder of Micrococcus lysodeicticus cells suspended in phosphate buffer pH 6.2 was quantitatively characterized in the temperature range from 34 to 52 degrees. The total value of the rate constant for heat inactivation of the cells equals 2.88 X 10(8) exp(-18360/RT) sec-1. The activation parameters of the process at 34 degrees are the following: delta H* = 17.7 kcal/mole; delta S* = 21.8 E. U.; delta F* = 24.4 kcal/mole. The effect of ethylene glycol, mannitol, dextran, polyvinyl alcohol (PVA) and polyethylene glycols with different molecular weights on the lysis rate and cell stability was studied. Polyvinyl alcohol was found to be the most effective stabilizer. At concentrations of about 10(-5) it enhances the thermostability of the cells threefold.     

Reaction of azide radicals with amino acids and proteins.

The azide radical N3 reacts selectively with amino acids, in neutral solution preferentially with tryptophan (k (N3 + TrpH) = 4.1 X 10(9) dm3 mol(-1s-1) and in alkaline solution also with cysteine and tyrosine (k(N3 + CyS-) = 2.7 X 10(9) dm3 mol-1s-1) and k(N3 + TyrO-) equals 03.6 X 10(9) dm3 mol-1s-1). Oxidation of the enzyme yADH by N3 involves primary attacks, mainly at tryptophan residues, and subsequent slow secondary reactions. N3-induced inactivation of yADH is likely to occur upon oxidation of tryptophan residues in the substrate binding pocket (58-TrpH and 93-TrpH) since the substrate ethanol although unreactive with N3, protects yADH and since elADH, which does not contain tryptophan in the substrate pocket, is comparatively resistant against N3-attack. N3 exhibits low reactivity with nucleic acid derivatives and it is inert towards aliphatic compounds such as methanol and sodium dodecylsulphate.