Benzyl alcohol penetration into micelles, dielectric constant of the binding site, partition coefficient and high-pressure squeeze-out

The absorbance maximum, lambda max, of a local anesthetic, benzyl alcohol, is shifted to longer wavelengths when solvent polarity is decreased. The shift was approximately a linear function of the dielectric constant of the solvent. This transition in electronic spectra according to the microenvironmental polarity is used to analyze benzyl alcohol binding to surfactant micelles. A facile method is devised to estimate the micelle/water partition coefficient from the dependence of lambda max of benzyl alcohol on surfactant concentrations. The effective dielectric constants of the sodium decyl sulfate, dodecyl sulfate and tetradecyl sulfate micelles were 29, 31 and 33, respectively. The partition coefficient of benzyl alcohol between the micelles and the aqueous phase was 417, 610 and 1089, respectively, in the mole fraction unit. The pressure dependence of the partition coefficient was estimated from the depression of the critical micelle concentration of sodium dodecyl sulfate by benzyl alcohol under high pressure up to 200 MPa. High pressure squeezed out benzyl alcohol molecules from the micelle until about 120 MPa, then started to squeeze in when the pressure was further increased. The volume change of benzyl alcohol by transfer from the aqueous to the micellar phase was calculated from the pressure dependence of the partition coefficient. The volume change, estimated from the thermodynamic argument, was 3.5 +/- 1.1 cm3.mol-1 at 298.15 K, which was in reasonable agreement with the partial molal volume change determined directly from the solution density measurements, 3.1 +/- 0.2 cm3.mol-1. Benzyl alcohol apparently solvates into the micelles close to surface without losing contact with the aqueous phase.     

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 (_max) (for tyrosine and phenylalanine) or the amplitude of the highest maximum (A_max) (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.     

Light-dependent nitration of bacteriorhodopsin

Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.     

Sensitive spectrophotometric determination of tetraphenylboron and tetraphenylarsonium with ethidium bromide.

Tetraphenylboron and tetraphenylarsonium ions have been determined spectrophotometrically by measuring the red shift in the absorbance maximum of ethidium on its reaction with tetraphenylboron at neutral pH. The present method permits the determination of nanomole quantities of these ions.     

A nonradioactive assay for poly(a)-specific ribonuclease activity by methylene blue colorimetry

A simple nonradioactive assay, which was based on the specific shift of the absorbance maximum of methylene blue induced by its intercalation into poly(A) molecules, was developed for poly(A)-specific ribonuclease (PARN). A good linear relationship was found between the absorbance at 662 nm and the poly(A) concentration. The assay conditions, including the concentration of methylene blue, the incubation temperature and time, and the poly(A) concentration were evaluated and optimized.     

Monomolecular films of bacteriochlorophyll and derivatives at an air-water interface: Surface and spectral properties

Monomolecular films of bacteriochlorophyll, bacteriopheophytin and 2-desvinyl-2-acetyl chlorophyll a were prepared and studied on aqueous subphases containing pH 7.8 buffer and 4·10⁻⁴ M ascorbate. These monolayers are mechanically stable in the dark and light at 15 °C. at surface pressures below about 18 dynes/cm the slope of the surface isotherm of bacteriochlorophyll is steeper than at pressures greater than 18 dynes/cm. The surface dipole moments of bacteriochlorophyll are less than half that reported for chlorophyll a. Compression of bacteriochlorophyll or bacteriopheophytin monolayers result in changes of their absorption spectra.

Compression of bacteriochlorophyll monolayers to 18 dynes/cm results in a shift of the pigment's red peak from 787 to 749 nm as well as the appearance of a new absorption maximum at 896 nm. Continued compression to 24 dynes/cm results in a slight decrease in peak height of the 794-nm maximum and further increase in the absorbance of the 896-nm maximum. With bacteriopheophytin the red maximum at 760 nm starts to shift when the film is compressed to a surface pressure of only 2 dynes/cm; further compression yields a new absorption maximum at 846 nm. Compression of a film of 2-desvinyl-2-acetyl chlorophyll a results in only a 10-nm shift of the absorption maximum at 690 nm.

An orientation of bacteriochlorophyll at an air-water interface is proposed that is different from that for chlorophyll a. Like chlorophyll a bacteriochlorophyll monolayers are closely packed, but different in that bacteriochlorophyll allows greater interaction between pigment molecules. In compressed monolayers bacteriochlorophyll appears to aggregate differently than in other model systems.     

Effect of alkaline PH on sunflower 11S Protein

The effect of alkaline PH on sunflower 1 1S Protein has been monitored by the techniques of ultracentrifugation, Polyacrylamide gel electroPhoresis, turbidity, viscosity, ultraviolet absorPtion sPectra and fluorescence sPectra. Both ultracentrifugation and Polyacrylamide gel electroPhoresis show the dissociation of the Protein with increase in PH. Turbidity values decrease with PH while viscosity increases. With increase in PH absorbance of the Protein solution increases and there is a red shift in the absorPtion maximum. Fluorescence quenching and a red shift in the emission maximum are also observed. Both dissociation and denaturation of the Protein occur. Analysis of turbidity, viscosity and fluorescence data suggests that aPParently denaturation follows dissociation.     

Absorbance and fluorescence properties of 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate bound to coupled and uncoupled Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Preincubation of skeletal muscle sarcoplasmic reticulum vesicles in the presence of the calcium chelator, [ethylenebis(oxyethylenenitrilo)tetraacetic acid] (EGTA), irreversibly uncouples calcium transport from ATP hydrolysis. Uncoupling cannot be explained by increased membrane permeability, but is associated with decreased capacity of the Ca2+-ATPase to bind noncatalytic, tightly bound ATP and ADP (Berman, M. C. (1982) Biochim. Biophys. Acta 694, 95-121). The effects of EGTA-induced uncoupling on absorbance and fluorescence properties of the bound ATP analog, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), have been studied under static and turnover conditions. Binding of 4.5-4.9 nmol of TNP-ATP/mg, as determined by absorbance difference titration, was relatively unaffected in the uncoupled state. TNP-ATP, bound to coupled vesicles during turnover, showed 6-8-fold enhanced fluorescence and a shift in the difference absorbance maximum from 510 to 493 nm, indicating increased hydrophobicity of the noncatalytic site. Turnover-dependent fluorescence enhancement was diminished by 60-70% in the uncoupled state, while the absorbance maximum wavelength shift was abolished. These data, correlating changes in the environment of the noncatalytic or regulatory nucleotide binding site on the Ca2+-ATPase with coupling activity, indicate that uncoupling is an intramolecular process, involving a ligand binding site on the ATPase, and that exclusion of H2O from the site occupied by noncatalytic nucleotides, during at least part of the catalytic cycle, is an event associated with energy transduction.     

Light-induced absorbance changes of carotenoids in brown algae

Light-induced absorbance changes were studied for brown algae with 23 species and a pronounced absorbance change around 563 nm was found in all algae examined. 3-(3,4-dichlorophenyl)-1,1-dimethylurea and gramicidin J suppressed the initial rate and the magnitude of the absorbance change. Carbonylcyanidem-chlorophenylhydrazone did not affect the initial rate but decreased the maximum level of the change. All thalli and the chloroplasts tested had an absorption band at around 540 nm due to fucoxanthin which accounted for about 70–90% of the total carotenoids in brown algae. It is proposed that the 563 nm-change is caused by the red shift of fucoxanthin responding to the light-induced change in the membrane potential of the thylakoid system.     

Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino)-cinnamaldehyde binding to the active site

Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry. This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm. Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data.     

Degradative inactivation of the peroxisomal enzyme, alcohol oxidase, during adaptation of methanol-grown Candida boidinii to ethanol.

Adaptation of methanol-grown C. boidinii to ethanol-utilization in non-growing cells resulted in decreased activity of the peroxisomal enzyme alcohol oxidase. Re-appearance of alcohol oxidase activity was dependent on protein synthesis de novo. Degradation of alcohol oxidase protein was shown to parallel the decrease in activity. Adaptation of methanol-grown cells to ethanol-utilization resulted in increased absorbance due to cytochromes and decreased absorbance due to flavoprotein. Decrease in alcohol oxidase activity was associated with loss of the flavin coenzyme, FAD, from the organisms and the appearance of flavins (FAD, FMN, riboflavin) in the surrounding medium. Electron microscopic observations showed that general degradation of whole peroxisomes rather than specific loss of crystalline cores (alcohol oxidase protein) occurred during the adaptation.     

Deamination of 4-Aminopyrimidine Nucleosides by Extracts of Rye Grass (Lolium perenne)

A deaminase specific for 4-aminopyrimidine nucleosides has been found in rye grass. Lolium perenne. The pH optimum, temperature stability, Km values and specificity was determined.

A new and more accurate method for following the course of deamination is reported. This method depends on the shift in maximum absorbance wavelength of the mixture containing the 4-aminopyrimidine nucleoside and its deaminated product.

     

Surface Plasmon Coupling Effect of Gold Nanoparticles with Different Shape and Size on Conventional Surface Plasmon Resonance Signal

The labeling strategy with gold nanoparticles for the conventional surface plasmon resonance (SPR) signal enhancement has been frequently used for the sensitive determination of small molecules binding to its interaction partners. However, the influence of gold nanoparticles with different size and shape on SPR signal is not known. In this paper, three kinds of gold nanoparticles, namely nanorods, nanospheres, and nanooctahedrons with different size, were prepared and used to investigate their effects on the conventional SPR signal at a fixed excitation wavelength 670 nm. It was found that the SPR signal (i.e., resonant angle shift) was varied with the shapes and sizes of gold nanoparticles in suspension at a fixed concentration due to their different plasmon absorbance bands. For gold nanorods with different longitudinal absorbance bands, three conventional SPR signal regions could be clearly observed when the gold nanorod suspensions were separately introduced onto the SPR sensor chip surface. One region was the longitudinal absorbance bands coinciding with or close to the SPR excitation wavelength that suppressed the SPR angle shift. The second region was the longitudinal absorbance bands at 624 to 639 and 728 to 763 nm that produced a moderate increase on the SPR resonant angle shift. The third region was found for the longitudinal absorbance bands from 700 to 726 nm that resulted in a remarkable increase in the SPR angle shift responses. This phenomenon can be explained on the basis of calculation of the correlation of SPR angle shift response with the gold nanorod longitudinal absorbance bands. For nanospheres and nanooctahedrons, the SPR angle shift responses were found to be particle shape and size dependent in a simple way with a sustaining increase when the sizes of the nanoparticles were increased. Consequently, a guideline for choosing gold nanoparticles as tags is suggested for the SPR determination of small molecules with binding to the immobilized interaction partners.     

A blue-green absorbing cross-linker for rapid photoswitching of peptide helix content

Azobenzene derivatives can be used to reversibly photoregulate secondary structure when introduced as intramolecular bridges in peptides and proteins. Here we report the design, synthesis, and characterization of a disubstituted N,N-dialkyl azobenzene derivative that absorbs near 480 nm in aqueous solution and relaxes with a half-life of approximately 50 ms at room temperature. The wavelength of maximum absorbance and the rate of thermal relaxation are solvent-dependent. An increase in the percentage of organic solvent leads, in general, to a blue shift in the absorbance maximum and a slowing of the relaxation rate. In accordance with the design, the thermal relaxation of the azobenzene cross-linker from cis to trans causes an increase in the helix content of one peptide where the linker is attached via cysteine residues spaced at i, i + 11 positions and a decrease in helix content of another peptide with cysteine residues spaced at i, i + 7. This cross-linker design thus expands the possibilities for fast photocontrol of peptide and protein structure.     

Comparison of kinetic and optical changes during guanidine denaturation of intestinal alkaline phosphatase

The modifications of the activity of calf intestinal Alkaline Phosphatase treated with moderate amounts of guanidinium chloride are compared with the conformational changes observed by ultraviolet absorbance and intrinsic fluorescence. The time course of catalytic and optical properties of the treated enzyme develops through two distinct steps: an instantaneous and a time-dependent one. The immediate effect of guanidine is to lower emission yield, to shift the emission maximum of the enzyme to longer wavelengths and to enhance the absorbance of the protein. The rapid conformational transition determines a paradoxical activation at low effector concentration (below 0.88 M) and an inhibition at higher amounts. The following marked decay of enzyme activity with time is related to spectroscopically detectable changes. Temperature influences both kinetic and structural aspects of the process and facilitates guanidine action.     

Non-covalent intercalative binding of 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene to DNA

When the benzo(a)pyrene diol epoxide (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is mixed into a DNA solution, a 10nm red shift in the absorption maximum of BPDE appears at 354nm which is due to a non-covalent intercalation complex. The major reaction pathway at this intercalation site is the hydrolysis of BPDE to its tetraol which is accompanied by a decrease in the absorbance and a shift from 354 to 353nm (the latter is due to intercalated tetraol). The non-covalent binding constants are approximately 8200M^?1 for BPDE and 3300M^?1 for the tetraol at 25°C, pH 7.0. Covalent adduct formation between BPDE and DNA occurs either at another, external binding site, or after some rearrangement of the intercalated BPDE, since covalent adducts display a 345nm absorption maximum (2nm red shift only).     

Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.     

Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate.

Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.     

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans-membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelength is obtained with valinomycin-induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.     

Concentration polarization phenomenon in the case of mechanical pressure difference on the membrane

We analyzed the transport of KCl solutions through the bacterial cellulose membrane and concentration boundary layers (CBLs) near membrane with pressure differences on the membrane. The membrane was located in horizontal-plane between two chambers with different KCL solutions. The membrane was located in horizontal-plane between two chambers with different KCL solutions. As results from the elaborated model, gradient of KCL concentration in CBLs is maximal at membrane surfaces in the case when pressure difference on the membrane equals zero. The amplitude of this maximum decreases with time of CBLs buildup. Application of mechanical pressure gradient in the direction of gradient of osmotic pressure on the membrane causes a shift of this maximum into the chamber with lower concentration. In turn, application of mechanical pressure gradient directed opposite to the gradient of osmotic pressure causes the appearance of maximum of concentration gradient in chamber with higher concentration. Besides, the increase of time of CBLs buildup entails a decrease of peak height and shift of this peak further from the membrane. Similar behavior is observed for distribution of energy dissipation in CBLs but for pressure difference on the membrane equal to zero the maximum of energy dissipation is observed in the chamber with lower concentration. We also measured time characteristics of voltage in the membrane system with greater KCl concentrations over the membrane. We can state that mechanical pressure difference on the membrane can suppress or strengthen hydrodynamic instabilities visible as pulsations of measured voltage. Additionally, time of appearance of voltage pulsations, its amplitude, and frequency depend on mechanical pressure differences on the membrane and initial quotient of KCl concentrations in chambers.