High-pressure stopped-flow fluorometry at subzero temperatures: application to kinetics of the binding of NADH to liver alcohol dehydrogenase

A stopped-flow apparatus operating in fluorescence mode over temperature and pressure ranges of +30 to -30 degrees C and 10(-3) to 2 kbar, respectively, is described. The system was interfaced on a special spectrofluorometer. Its general design is an improvement of the previous instrument (C. Balny, J. L. Saldana, and N. Dahan, (1984) Anal. Biochem. 139, 178-189) in that the observation chamber and the driving mechanism have been modified. The application of the method to kinetics of the binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures and as a function of hydrostatic pressure is described.     

Refolding of beta-lactoglobulin studied by stopped-flow circular dichroism at subzero temperatures

Refolding of bovine beta-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%-buffer 55% at -15 degrees C, the isomerization rate from the kinetic intermediate rich in alpha-helix to the native state is approximately 300-fold slower than that at 4 degrees C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At -28 degrees C, we observed at least three phases; the fastest process, accompanied by an increase of alpha-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of alpha-helix content with the rate of 2 s(-1), and the third phase, accompanied by a decrease of alpha-helix content. This last phase, corresponding to the isomerization process at -15 degrees C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid alpha-helix formation and their concurrent collapse are common even in proteins rich in beta-structure in their native forms.     

Photodynamic hemolysis at low temperatures

It is shown that photodynamic hemolysis may occur at –79°C. if the erythrocytes are suspended in a solution containing 70 per cent glycerol which prevents hemolysis by freezing; but that there is no hemolysis under the same conditions at –210°C. At the higher temperature the viscosity of the solution is still low enough to permit appreciable movement of molecules, whereas at the lower temperature the molecules must be virtually immobile. The findings are compatible with the idea that the dye molecule acts in a cycle, bringing about successive oxidations by O₂ molecules, as has been shown for photodynamic hemolysis at room temperature. The assumption of a combination between dye, O₂, and substrate does not explain photosensitized hemolysis in the semi-solid state. The mechanism of photosensitized oxidation by O₂ is discussed.     

NMR and protein folding: equilibrium and stopped-flow studies.

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped-flow device incorporated into the NMR spectrometer.     

Kinetic study on myoglobin refolding monitored by five optical probe stopped-flow methods

The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 × 10² s^?1, (4.5–9.3) S^?1, and (2–5) × 10^?3 s^?1. In the fastest phase, a substantial amount of secondary structure (40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group. © 1994 Wiley-Liss, Inc.     

A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.     

Fluorescence stopped-flow study of the interaction of alkylpyridinium salts and pyrene-substituted fatty acids with lecithin vesicles

The migration of alkylpyridinium surfactants and pyrene-substituted fatty acids between vesicles has been studied. The results are in accord with the assumption of diffusion-controlled processes. Changes in relaxation time with charge type, degree of ionization, pH, and alkyl chain length of the migrating species are investigated and discussed.The presence of a slower relaxation type, apparently due to a transmembrane transport was established in the case of the pyridinium surfactants, but could not be found for pyrene-substituted fatty acids. Fluorescence-quenching studies indicate that these are present at both vesicle interfaces.     

Minnows become nocturnal at low temperatures

Juveniles of several species of salmonid become almost exclusively nocturnal in winter. Their diel activity schedule is determined mainly by temperature, with the fish seeking shelter increasingly during the day as the temperature drops. Through controlled laboratory experiments, it is demonstrated that a comparable response is found in another stream-dwelling species, the minnow Phoxinus phoxinus . Daytime observations showed that fish were found hiding in refuges on only 20% of occasions at 13.2° C but on over 75% of occasions at 6.6° C. In contrast, the minnows remained in the water column and used the refuges rarely at night irrespective of water temperature. As a consequence their activity became increasingly nocturnal as temperature dropped. It is suggested that this may be an adaptation to avoid diurnal predators.     

Bacterial gene expression at low temperatures

Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.     

Microbial lipolysis at low temperatures.

It was found that lipase production during the growth of Pseudomonas fluorescens was not a function of the total number of bacteria. The optimal temperatures for bacterial growth and lipase production were determined as 20 and 8 degrees C, respectively. The lipolytic activity was studied in emulsions of olive oil at temperatures ranging from +8 to -30 degrees C. After an initially rapid lipolysis, the reactions retarded at different levels depending on storage temperature. Transference to a higher temperature resulted in a resumed lipolysis. Also, at low temperatures, lipolysis was studied as a function of water activity and was found to occur in dehydrated substrates.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.     

A stopped-flow fluorescence study of the native and modified lysozyme

The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.     

The feeling of dampness at low temperatures

The widely held view that in cold weather one feels colder when the atmosphere is damp than when it is dry was investigated on clothed subjects. Subjective coldness and dampness, determined by a panel of six people who walked about half a mile in the open in winter, were compared with measurements of temperature, relative humidity, and other meteorological variables. The panellists comments showed that when the weather is cold, people think it is damp when the sky is overcast, irrespective of relative humidity. However, when this factor has been allowed for there still appears to be some correlation between subjective dampness and relative humidity. Relative humidity does appear to have an effect on how cold one feels at low temperatures, but only to a limited extent, and in the opposite direction to popular belief.     

Growth of mesophilic methanotrophs at low temperatures

The optimal growth of mesophilic methanotrophic bacteria (collection strains of the genera Methylocystis, Methylomonas, Methylosinus, and Methylobacter) occurred within temperature ranges of 31-34 degrees C and 23-25 degrees C. None of the strains studied were able to grow at 1.5 or 4 degrees C. Representatives of six methanotrophic species (strains Mcs. echinoides 2, Mm. methanica 12, Mb. bovis 89, Mcs. pyriformis 14, Mb. chroococcum 90, and Mb. vinelandii 87) could grow at 10 degrees C (with a low specific growth rate). The results obtained suggest that some mesophilic methane-oxidizing bacteria display psychrotolerant (psychrotrophic) but not psychrophilic properties. In general, the Rosso model, which describes bacterial growth rate as a function of temperature, fits well the experimental data, although, for most methanotrophs, with symmetrical approximations for optimal temperature.