Claude Balny

High pressure effects on protein structure and function

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

Effects of high pressure on proteins

Abstract The recent interest of the food industry in the high‐pressure processing of food materials as an alternative or in addition to temperature treatment requires fundamental studies on the pressure‐temperature behavior of macromolecular food constituents such as proteins. In this paper we review some basic knowledge on the effects of high pressure on proteins. These effects are reversible or nonre‐versible and include changes in intra‐ or intermolecular interactions (noncovalent bonds), in conformation and in solvation. In general, reversible effects are observed below 1–2 kbar (e.g., dissociation of polymeric structures into subunits). Above 2 kbar, nonreversible effects may include complete inactivation of enzymes and denaturation of proteins (unfolding of monomeric proteins, aggregation, and gelation phenomena). Particular attention is directed to pressure denaturation, a complex phenomenon depending on protein structure, on pressure range, and on other external parameters, for example, temperatur...

Exploiting the effects of high hydrostatic pressure in biotechnological applications

Abstract Applying hydrostatic pressure to biological systems and processes can alter their characteristics. In addition to its use as a basic research tool for investigating the kinetics and thermodynamics of biological systems at the molecular level, the application of pressure is also being used to modify the properties of biological materials to preserve or improve their qualities. This article reviews the principles underlying the observed effects of applied pressure on biological systems, and discusses current and potential application of pressure in biotechnological processes.

Application of high hydrostatic pressure for increasing activity and stability of enzymes

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure.

Reversible changes of the wheat γ46 gliadin conformation submitted to high pressures and temperatures

The structure of the wheat gamma 46 gliadin was investigated, in aqueous solutions, under high pressure or temperature by the use of ultraviolet and fluorescence spectroscopic techniques. We found that high pressure (above 400 MPa) induces a change in the protein conformation that results in a decrease of the polarity of the environment of aromatic amino acids. This new conformation was able to bind the hydrophobic probe, 8-anilino-1-naphtalene-sulfonic acid (ANS), indicating an increase in the gliadin surface hydrophobicity. Thermodynamic parameters of this conformational change were measured and infrared spectroscopy studies were used to probe the potential secondary structure modifications. The high stability of gamma 46 gliadin could be related to its elastic character, as the observed changes were always found to be reversible.

High-pressure as a tool to study some proteins’ properties: conformational modification, activity and oligomeric dissociation

Abstract Hydrostatic pressure, as temperature, constitutes an efficient physical parameter to modify equilibrium and rate of biological processes. In this review, we will not present all the implications of high-pressure, but we will focus on proteins’ structural conformational modification, unfolding, oligomeric or protein aggregates dissociation and enzymatic activity. To this aim, some optical methods that were used in association with high-pressure to study protein structures (i.e. fourth derivative absorbance spectroscopy, fluorescence spectroscopy, infrared spectroscopy) and protein activities (kinetics measurements: stopped-flow and pressure jump methods) will be described in details. Some applications of these methods will be given including effects on proteins of food interest.

Some recent aspects of the use of high-pressure for protein investigations in solution

Abstract Proteins play a key role in the metabolism of living organisms as, for example, catalysts (enzymes), carriers or receptors of various molecules. These marginally stable biopolymers of amino acids can be perturbed in their activity by pressure, temperature and other environmental variables as organic solvents. Changing environmental conditions can induce metabolism dysfunctions. On isolated systems, i.e. purified proteins, high pressure and other perturbing variables can be used as tools for investigation of protein structure/activity relationships and enzyme mechanisms. The elementary basic physical mechanisms of the action of high pressure upon proteins are stated in the first part of this review. This is followed by a section devoted to technical aspects, including methods for generation of high pressure, and some recent developments, namely stopped-flow spectrometry and electrophoresis under high pressure. Then, the use of pressure as a tool for investigation of enzyme mechanisms and for study...

High-pressure biotechnology in medicine and pharmaceutical science

High-pressure (HP) biotechnology is an emerging technique initially applied for food processing and more recently in pharmaceutical and medical sciences. Pressure can stabilize enzymes and modulate both their activity and specificity. HP engineering of proteins may be used for enzyme-catalyzed synthesis of fine chemicals, pharmaceuticals, and production of modified proteins of medical or pharmaceutical interest. HP inactivation of biological agents is expected to be applicable to sterilization of fragile biopharmaceuticals, or medical compounds. The enhanced immunogenicity of some pressure-killed bacteria and viruses could be applied for making new vaccines. Finally, storage at subzero temperatures without freezing is another potential application of HP for cells, animal tissues, blood cells, organs for transplant, and so forth.

Fourth derivative UV-spectroscopy of proteins under high pressure I. Factors affecting the fourth derivative spectrum of the aromatic amino acids

A tunable fourth derivative UV absorbance method based on a variable spectral shift has been developed and compared to the Savitzky-Golay method and the analytical derivative. The parameters of the method were optimised for the analysis of the UV absorbance spectra of the aromatic amino acids to quantify the effect of decreasing solvent polarity on their fourth derivative spectra. The wavelength of the highest maximum (λmax) (for tyrosine and phenylalanine) or the amplitude of the highest maximum (Amax) (for tryptophan), were shown to depend linearly on the dielectric constant of the solvent, ranging from water to cyclohexane. The only effect of pressure in the 1 to 500 MPa range is a small decrease in the fourth derivative amplitude. This method appears therefore as a suitable tool to evaluate changes of the dielectric constant in the vicinity of the aromatic amino acids in proteins which undergo pressure induced structural changes.

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.