An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.     

An osmometer for aqueous biopolymer solutions

A sensitive osmometer for the study of the osmotic pressure of aqueous protein solutions is presented. Equilibrium is reached within 1 to 2 h and is reproducible to about 1 N.m-2 (= 0.01 cm H2O). The method allows the determination of dissociation constants of proteins down to 1 x 10(-8) M.     

An infrared study on the conformation of poly-L-proline in concentrated aqueous salt solutions

The infrared absorption of poly-L -proline in concentrated aqueous salt solutions was measured in the fundamental region. Of primary interest were the carbonyl absorption of the peptide linkage and the methylene C–H bending absorption of the pyrrolidine ring. These spectral regions each show an additional component in the concentrated salt solutions. Using the position of the absorptions of poly-L -proline I (cis) and II (trans) as models, we conclude that both cis–trans linkages are present in the peptide in salt solutions. Increasing the temperature shifts the equilibrium slightly in favor of cis.     

Vibrational spectroscopic study of glutathione complexation in aqueous solutions

A spectroscopic study of glutathione (GSH) and glutathione disulfide (GSSG) has been performed using Fourier-transformed infrared absorption and Raman scattering in order to pinpoint the sites of complexation of these two species with water and particularly with H2O2. Molecules of GSH and GSSG were studied in KBr pellets, and in aqueous solutions of H2O, D2O, and H2O with H2O2 (1 mol L(-1)) to characterize the specific influence of the solvent molecules. A time-resolved Raman study was performed for GSH/H2O2, in aqueous solution at 1:1 molar ratio in order to observe the formation of GSSG and to discuss the mechanism of this redox reaction.     

An experimental study of mitosis

A comparison of colchicine-treated and normal root tips of onion provides information on the role of the spindle in mitosis, and clearly indicates the value of colchicine in the analysis of karyotypes. An improved Feulgen-squash technique is recommended, with which high quality preparations of chromosomes can be obtained as a matter of routine. This cytological exercise requires little equipment and is suitable for undergraduate courses and advanced classes in schools.     

An experimental study of the dielectric dispersion of solutions of cytochrome c at medium ionic strengths

In this paper, the results are presented of measurements of the dielectric dispersions of horse heart cytochrome c molecules in various buffers. The data are fitted to the Cole-Cole relaxation model. The influence of the concentration and the ionic strength on the parameters that result from the Cole-Cole model is determined. The measured data are compared with calculations based on the model presented previously. Good agreement is found between the model and the observed data.     

Kerr effect study of the aqueous solutions of three globular proteins

A Kerr effect study is reported in which measurements have been made on the magnitudes of both the steady maxima and the decays of the birefringence of solutions of ovalbumin, bovine γ-globulin, and β-lactoglobulin. For each protein, results are presented on solutions covering the concentration range of 0.3–1.7 g./100 ml. in order to obtain by extrapolation, values of the specific Kerr constant K_sp, and the birefringence relaxation time τ_{25, w} at zero concentration. The relaxation times thus obtained for ovalbumin (18.3 nsec.) and γ-globulin (157 nsec.) have been shown to be compatible with molecular models and dimensions presented in the literature. All experiments showed the need for careful extrapolation to zero concentration if reliable parameters are to be obtained: for example a 1% solution of ovalbumin or l.5% solution of γ-globulin, would give values for τ which are 50% too high when compared with the true value at infinite dilution. The gradual fall in τ for γ-globulin as the pH was lowered from 6.7 to 3.0 was also studied for three solvents. Fisher's generalized model for the arrangement of the polar residues around the outside of a globular protein has been developed to account for ellipsoidal particles and has been used to demonstrate the suitability and usefulness of this treatment in predicting the conformation and dimensions of these proteins. Rather unusual birefringence traces for β-lactoglobulin were obtained, which may indicate the dissociation of aggregates, or of the parent molecule into its subunits, under the influence of strong electric fields.     

A kinetic study of D-glucose oxidation by bromine in aqueous solutions

The kinetics of the oxidation of D-glucose to D-gluconic acid by bromine in aqueous solution were studied using potentiometric techniques and theoretical considerations of complex bromine-bromide-pH equilibria. The pH has a strong influence on reaction rate. At pH < 8 the reaction is very slow, while in the pH range pH 8-9.5 the reaction is sufficiently fast and seems optimal for the reaction. The proposed active species at that pH region is hypobromous acid. At pH > 9.5, the reaction is further accelerated due to the formation of hypobromite. The proposed kinetics expression for gluconic acid formation, based on the determined kinetic parameters at pH 9.24, is of the form dc(GA)/dt = 160c(2)(G)c(o)(HOBr)c(o)(H(+)c(o)(Br)     

Thermodynamic and spectroscopic study of intermolecular association in aqueous dipeptide solutions

Peculiarities of the concentration dependences of apparent molar heat capacities and volumes of aqueous solutions of dipeptides were considered. Possible interpretation of the character of these dependences on the basis of Gibson and Sheraga's model of solvation shell were shown. Three concentration regions corresponding to the existence of various molecular forms of dipeptides in solution were marked out.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.     

An efficient petiole-feeding bioassay for introducing aqueous solutions into dicotyledonous plants

Introducing bioactive molecules into plants helps establish their roles in plant growth and development. Here we describe a simple and effective petiole-feeding protocol to introduce aqueous solutions into the vascular stream and apoplast of dicotyledonous plants. This 'intravenous feeding' procedure has wide applicability to plant physiology, specifically with regard to the analysis of source-sink allocations, long-distance signaling, hormone biology and overall plant development. In comparison with existing methods, this technique allows the continuous feeding of aqueous solutions into plants without the need for constant monitoring. Findings are provided from experiments using soybean plants fed with a range of aqueous solutions containing tracer dyes, small metabolites, radiolabeled chemicals and biologically active plant extracts controlling nodulation. Typically, feeding experiments consist of (i) generating samples to feed (extracts, solutions and so on); (ii) growing recipient plants; (iii) setting up the feeding apparatus; and (iv) feeding sample solutions into the recipient plants. When the plants are ready, the feeding procedure can take 1-3 h to set up depending on the size of experiment (not including preparation of materials). The petiole-feeding technique also works with other plant species, including tomato, chili pepper and cabbage plants, as demonstrated here.     

A study of diluted aqueous solutions of humic acids by scanning microcalorimetry

It has been found by reversed-phase chromatography that humic acids obtained from vermicomposts of different duration of vermicomposting consist of a hydrophilic and a hydrophobic fractions, the hydrophobic fraction having a substantially lower content of charged, probably carboxylic, groups. A change in the sign of the temperature dependence of the heat capacity of diluted aqueous solutions of humic acids at approximately 58 degrees C has been found by differential scanning microcalorimetry, which indicates an increase in the hydration of hydrophobic groups. A jumpwise increase in heat capacity in the temperature range from 86 to 90 degrees C was also found, which is due likely to the hydration of hydrophobic groups in the interior of "micelles", due to the "devitrification" of the hydrophobic nucleus of micelle-like structures. It was shown that increasing the duration of vermicomposting leads to an increase in the relative content of the hydrophobic fraction of humic acids and in the cooperativity of the thermodynamic transition, which manifests itself in a jump of heat capacity, which probably results from the increase in the "micelle" size.