The secondary structure of human plasma fibronectin: conformational changes induced by acidic pH and elevated temperatures; a circular dichroic study

The secondary structure of native human plasma fibronectin, based on circular dichroic spectra, has been estimated to contain 79% beta sheet and 21% beta turn structures (Osterlund, E., Eronen, I., Osterlund, K. and Vuento, M. (1985) Biochemistry 24, 2661-2667). In this work changes in the secondary structure of the protein molecule are followed as a function of different temperatures and pH values by using circular dichroic spectroscopy in far- and near-ultraviolet regions. Conformational changes are reversible when raising the temperature quickly to 55 degrees C, and then cooling slowly to 20 degrees C. A few percent of alpha-helix is apparent, when the temperature is raised to 58.5 degrees C, but only about 9% random coil is formed, when the temperature is raised up to 70 degrees C. The largest conformational change is taking place, when fibronectin samples are heated from 57 to 58.5 degrees C. According to this study more than 90% of the secondary structure of the fibronectin molecule is preserved throughout the whole temperature range studied from 20 to 70 degrees C, and this is a fact even at pH as low as 3.0, when samples are fresh and not denatured by preparative procedures.     

Nuclear-magnetic-resonance studies of ferrocytochrome c. pH and temperature dependence

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 degrees C and over the temperature range 4 degrees C to 97 degrees C at pH 7.0. The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upfield. Tyr-48 and Phe-46 were found to be relatively immobile whilst a region of the protein close to Ile-57 was found to be relatively flexible.     

Aspects of morphine chemistry important to persons working with cold-blooded animals, especially fish

The relative amounts of the different forms of morphine, and many other pharmacologic agents, depend on temperature and pH. Some forms are more efficacious because they are uncharged and can penetrate lipid membranes more easily than the charged forms. Persons who administer pharmacologic agents to ectotherms (that is, cold-blooded animals) should consider the effect of temperature on the relative amounts of the different forms of drugs. For example, the fraction of morphine present in the uncharged form is twice as high in a fish or frog at 5 degrees C as in a mammal at 37 degrees C. Moreover, because the pH of blood, plasma, and tissues of ectotherms is higher when they are held at lower temperatures, the combined effect of temperature and pH on the speciation of pharmacologic agents also should be considered. In addition, the total solubility of morphine and other pharmacologic agents depends on temperature and pH. The purpose of this overview is to describe how temperature and pH influence the solubility and speciation of morphine.     

Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.     

Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin

A 42-kDa gelatin binding fragment of human plasma fibronectin was labeled with fluorescein and its fluid-phase interaction with gelatin was investigated. At 25 degrees C in 0.1 M Tris, 0.15 M NaCl, pH 7.3, a dissociation constant, Kd = 0.6 microM, was obtained from the dependence of fluorescence polarization on gelatin concentration. An identical value was obtained for the unlabeled fragment by competition. Binding was unaffected by higher concentrations of NaCl up to 1.0 M, but increased as much as 20-fold at low ionic strength. The dependence of Kd on temperature revealed that dissociation of the complex is accompanied by an increase in entropy. Thus, the interaction is not dominated by either hydrophobic or electrostatic forces; an important role for hydrogen binding is proposed.     

Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study

Analysis of parameters governing heparin binding to fibronectin indicates that heparin binding is a necessary, but insufficient, condition for fibronectin cryoprecipitation. Heparin binding to fibronectin is a rapid, readily reversible event which can occur under several conditions which prohibit fibronectin cryoprecipitation. While cryoprecipitation of fibronectin is abolished at temperatures in excess of 10 degrees C, appreciable heparin binding to fibronectin does occur even at 40 degrees C. While increasing ionic strength and pH inhibit both heparin binding and cryoprecipitation of fibronectin, heparin binding can still occur at high ionic strengths and pH values which completely abolish cryoprecipitation. Scatchard analysis of fluorescent polarization data reveals a biphasic heparin binding curve with high and low affinity Kd values of 3.5 X 10(-8) and 10(-6) M, respectively. In contrast to heparin binding, fibronectin aggregation is a cooperative phenomenon. Fibronectin cryoprecipitation is greatly reduced at temperatures above 10 degrees C, at pH values above pH 10, and at ionic strengths above 0.3 M. Thus, heparin binding and protein aggregation are separate events which occur during fibronectin cryoprecipitation. Results obtained here via fluorescence polarization in conjunction with other physical measurements suggest that a decrease in flexibility of the fibronectin molecule is associated with the protein aggregation step of cryoprecipitation. The role of heparin in the mechanism of fibronectin cryoprecipitation is discussed.     

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.     

Physico-chemical aspects of solubility of myosin in aqueous media

Solubility of fish (Labio rohita) myosin has been studied at varying temperatures in presence of various inorganic salts like NaCl, KCl, NaBr, Na2SO4, KI, and organic solutes like sucrose and urea. The effect of pH on the solubility has also been studied both in absence and presence of NaCl. Thermal denaturation temperatures of myosin in presence of NaCl, KCl, NaBr and Na2SO4 were found to be 40 degrees, 40 degrees, 45 degrees and 50 degrees C respectively. Thermodynamic parameters like changes in standard free energy (delta G degrees), enthalpy (delta H degrees) and entropy (delta S degrees) for precipitation of myosin from solution phase to gel phase have been evaluated and the physico-chemical aspects have been critically discussed. The average delta G degrees for gel formation varied only between -30 and -40 kJ/mole of myosin, although the nature of solutes, temperature and folding state of protein have been grossly altered. A compensation effect has also been exhibited from the linear plot of average values of delta H degrees against T delta S degrees for various solutes.     

Kinetic modeling of lipase catalyzed hydrolysis of (R/S)-1-methoxy-2-propyl-acetate as a model reaction for production of chiral secondary alcohols

The Candida antarctica lipase B catalyzed kinetic resolution of (R/S)-1-methoxy-2-propyl-acetate was studied as a model system for the biocatalytic production of chiral secondary alcohols. For this purpose, a kinetic model is proposed involving both enantiomers of this reaction using model discrimination and parameter identification. Starting from a ping-pong bi-bi mechanism, a simplified model with sensitive parameters was derived for the R- and S-enantiomer, respectively. It was validated at pH 7.0, using time-course measurements at varying temperatures (30-60 degrees C) and initial substrate conditions (0.05-1.5 M). This model was then used for mechanistic interpretation of the kinetic resolution on a biochemical level. The effect of temperature on kinetic parameters and enantiomeric ratio was investigated and compared to findings from the field of molecular modeling to obtain a better understanding of the reaction system for process design. Values of 21.2 and 9.7 kJmol-1 were determined for the enthalpic (DeltaR-S DeltaH ++ degrees) and the entropic (-T x DeltaR-S DeltaS ++ degrees) contribution of the difference in transition state energy of both enantiomers at 30 degrees C. High enantiomeric ratio's (E of 47-110) especially at lower temperatures, in addition to enzyme activity at a wide pH range, indicate this biotransformation is a promising example for the industrial production of chiral secondary alcohols.     

Aggregation and fibril formation of plasma fibronectin by heparin

The precipitation of plasma fibronectin by heparin in dependence on various parameters was investigated. Rising heparin concentration augmented the precipitates up to a maximum beyond which precipitation decreased. Yields close to 80% were obtained at low temperatures, but some precipitation was observed at 37 degrees C as well. Insolubilization was considerably dependent on the ionic strength, indicating that electrostatic forces play a major role in the aggregation of fibronectin. Calcium already prevented precipitation by heparin at low concentrations. If precipitation was performed on hydrophobized glass cover slides, the formation of fibrils visible by phase-contrast microscopy was observed. On hydrophilic surfaces amorphous precipitates were generally obtained, most likely due to trapping of aggregates by adsorption prior to their arrangement to fibrils. The results are discussed on the basis of a model assuming that heparin induces a conformational rearrangement of plasma fibronectin so that masked binding sites responsible for self-association become exposed.     

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.     

Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.     

The oxygen uptake of Sarotherodon niloticus L. and the oxygen binding properties of its blood and hemolysate (Pisces: Cichlidae)

The oxygen consumption of Sarotherodon niloticus L. was found to decline below a critical oxygen concentration of about 2 mg O2/l. An important influence of CO2 on the oxygen affinity of whole blood was observed at all temperatures between 20 and 35 degrees C for gas mixtures containing 5.6% CO2. Purified hemolysate showed extremely high oxygen affinities (p50 = 1.08 mmHg at pH 8.2 and 20 degrees C). Low cooperativity was observed at all temperatures from 20 to 35 degrees C, and pH values between 6.5 and 8.2. The Bohr effect proved to be important at pH values lower than pH 7.5 (phi = delta log P50/delta pH = -0.58 between pH 6.5 and 7.0 at 35 degrees C). The oxygen affinities show high thermal sensitivity without a marked pH influence (delta H value for overall oxygenation at pH was -71.7 kJ/mol). The obtained results are interpreted as adaptations to diurnal variations in ambient temperature and oxygen availability.     

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.     

Structural transitions of bovine serum albumin detected during scanning calorimetry

The calorimetry studies of temperature dependence of bovine serum albumin heat capacity for the temperature interval 20-150 degrees C and pH value varying from 4 to 9 were carried out. It is shown that in the pH range considered four types of denaturation curves differing in quantity and temperatures of endothermic pikes exist. The different types of the melting curves correspond to different types of protein domain structure. At 110-120 degrees C and pH greater than or equal to 5.6 the high temperature maximum is shown to exist. The later is supposed to be due not to BSA denaturation process but to cooperative destruction of postdenaturation remnants of the secondary protein structure.     

Thermodynamics of complex formation between nicotinamide adenine dinucleotide and pig skeletal muscle lactate dehydrogenase.

Formation of the binary complex between the reduced coenzyme nicotinamide adenine dinucleotide (NADH) and pig skeletal muscle lactate dehydrogenase (LDH, EC 1.1.1.27) has been investigated by calorimetric and equilibrium dialysis techniques in 0.2 M potassium phosphate buffer (pH 7.0) at various temperatures. Analysis of thermal titration curves at two temperatures (25 and 31.5 degrees) shows that the experimental enthalpy data can be rationalized assuming four independent and equivalent binding sites for the tetrameric enzyme. Binary complex formation is characterized by a negative temperature coefficient, delta cp, of the binding enthalpy, which amounts to -1300 plus or minus 53 cal/(deg mol of LDH) in the temperature range of 5-31.5 degrees. Despite the slightly smaller standard deviation resulting when polynomial regression analysis of the second degree is applied to the temperature dependence of the enthalpy values, binding enthalpies seem to be adequately represented in the temperature range studied by the equation delta H = -1.3T + 2.3, kcal/mol of LDH, T referring to the temperature in degrees C. By combination of the results obtained from equilibrium dialysis and calorimetric studies a set of apparent thermodynamic parameters for binding of NADH to LDH in 0.2 M potassium phosphate buffer at pH 7 has been established.     

pH-dependent binding of peroxidase-antiperoxidase /PAP/ immune complex to Fc receptors of macrophages

Rat peritoneal macrophages were incubated at 4 degrees C in media of varying pH-s containing rat peroxidase-antiperoxidase /PAP/ immune complex. Two binding maxima were found at pH 5.5 and 7.0, resp. There was a sharp decrease in binding between 5.5 and 5.0. The peak at pH 7.0 was found to be trypsin-sensitive.     

Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua)

Uracil-DNA glycosylase (UDG; UNG) has been purified 17000-fold from Atlantic cod liver (Gadus morhua). The enzyme has an apparent molecular mass of 25 kDa, as determined by gel filtration, and an isoelectric point above 9.0. Atlantic cUNG is inhibited by the specific UNG inhibitor (Ugi) from the Bacillus subtilis bacteriophage (PBS2), and has a 2-fold higher activity for single-stranded DNA than for double-stranded DNA. cUNG has an optimum activity between pH 7.0-9.0 and 25-50 mM NaCl, and a temperature optimum of 41 degrees C. Cod UNG was compared with the recombinant human UNG (rhUNG), and was found to have slightly higher relative activity at low temperatures compared with their respective optimum temperatures. Cod UNG is also more pH- and temperature labile than rhUNG. At pH 10.0, the recombinant human UNG had 66% residual activity compared with only 0.4% for the Atlantic cUNG. At 50 degrees C, cUNG had a half-life of 0.5 min compared with 8 min for the rhUNG. These activity and stability experiments reveal cold-adapted features in cUNG.     

Temperature effects on cyclic AMP accumulation in cultured fibroblasts

The kinetic parameters that determine the accumulation of cAMP in WI-38 cells stimulated with prostaglandin E1 have been determined at 37 degrees C and at lower temperatures. For desensitized cells, a reduction of temperatures from 37 degrees to 25 degrees C reduced both rate of synthesis and rate of elimination of cAMP by about 40%. The steady-state accumulation was, therefore, about the same at both temperatures. The extent of desensitization was also shown to be comparable at the two temperatures. It can be inferred that there was appreciable desensitization at 4 degrees C after a period of stimulation of less than one hour. This is contrasted with the behavior of C6-2B glioma cells at the same temperature. Escape of cAMP through the plasma membrane showed a greater temperature dependence than any of the other processes concerned with cAMP accumulation.     

A weaker gelatin-binding affinity and increased glycosylation of amniotic fluid fibronectin than plasma fibronectin

Human amniotic fluid fibronectin had different carbohydrate moieties from plasma fibronectin. Nearly 90% of glycopeptides released from amniotic fluid fibronectin was not bound by concanavalin A-Sepharose, whereas 75% of glycopeptides from plasma fibronectin was bound. Amniotic fluid fibronectin showed a significantly lower gelatin-binding affinity than plasma fibronectin at 25 degrees C. When the incubation temperature was lowered to 4 degrees C, no significant difference in this activity was found. Cell-attachment promoting activity of the two fibronectins was not significantly different.