Hydrophilic Microenvironment Research Articles

Effects of i-propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence.

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady-state, time-resolved, and stopped-flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild-type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so-called "lid," an alpha-helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild-type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild-type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (tau(1) = 0.5--1.2 ns) for the wild-type TLL nor in the longer fluorescence lifetime component (tau(2) = 4.8--6.0 ns) in the wild-type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for tau(1) increased significantly, from 0.43 to 1.5 ns. The shorter correlation time phi(1) of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r(infinity) the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped-flow fluorescence spectroscopy measurements suggested the lid to open within approximately 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.

Emulsifying Potency of New Amino Acid-Type Surfactant. (1). O/W Emulsions.

Oil-in-water (O/W) emulsions were prepared using a new amino acid-type emulsifier (surfactant), N-[3-lauryloxy-2-hydroxypropyl]-L-arginine L-glutamate (C12HEA-Glu), and the emulsifying potency of C12HEA-Glu was examined in terms of dispersibility, droplet size, interfacial tension, viscosity and hydrophilic microenvironment of droplet surface, whose emulsifying potency was compared with that of general amphoteric and nonionic surfactants. The effects of the constituent oils on the stability of emulsions were also examined. The emulsifying potency of C12HEA-Glu was greater than that of amphoteric N-lauryl-N, N-dimethyl-α-betaine and nonionic polyoxyethylene(10) laurylether, especially, the stability of emulsions containing oleic acid (OA), (C12HEA-Glu/OA/water system), was greatest, while the stability of emulsions containing octane (OC), (C12HEA-Glu/OC/water system), was inferior. OA possesses a hydrophilic carboxyl group and the viscosity of OA is higher than that of OC. The higher visco-elasticity of OA droplet surface and the formation of hydration layer on the droplet contributed to the greater stability of OA emulsions, thereby raising the stability of emulsions for the system C12HEA-Glu/OA/water in spite of the small | zeta-potential | (insufficiently electrostatic repulsion) of droplets.

A Self-Consistent, Microenvironment Modulated Screened Coulomb Potential Approximation to Calculate pH-Dependent Electrostatic Effects in Proteins

An improved approach is presented for calculating pH-dependent electrostatic effects in proteins using sigmoidally screened Coulomb potentials (SCP). It is hypothesized that a key determinant of seemingly aberrant behavior in pKa shifts is due to the properties of the unique microenvironment around each residue. To help demonstrate this proposal, an approach is developed to characterize the microenvironments using the local hydrophobicity/hydrophilicity around each residue of the protein. The quantitative characterization of the microenvironments shows that the protein is a complex mosaic of differing dielectric regions that provides a physical basis for modifying the dielectric screening functions: in more hydrophobic microenvironments the screening decreases whereas the converse applies to more hydrophilic regions. The approach was applied to seven proteins providing more than 100 measured pKa values and yielded a root mean square deviation of 0.5 between calculated and experimental values. The incorporation of the local hydrophobicity characteristics into the algorithm allowed the resolution of some of the more intractable problems in the calculation of pKa. Thus, the divergent shifts of the pKa of Glu-35 and Asp-66 in hen egg white lysozyme, which are both about 90% buried, was correctly predicted. Mechanistically, the divergence occurs because Glu-35 is in a hydrophobic microenvironment, while Asp-66 is in a hydrophilic microenvironment. Furthermore, because the calculation of the microenvironmental effects takes very little CPU time, the computational speed of the SCP formulation is conserved. Finally, results from different crystal structures of a given protein were compared, and it is shown that the reliability of the calculated pKa values is sufficient to allow identification of conformations that may be more relevant for the solution structure.

Preparation, characterization and application of immobilized carboxypeptidase A

Bovine carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.17.1) was covalently attached to polyacrylamide beads and to silica-based supports, and also adsorbed on polyethylene terephthalate. The highest immobilized activity (125 U g −1 solid) was achieved when a polyacrylamide bead support (Akrilex C) possessing carboxylic functional groups activated by a water-soluble carbodiimide was used. The catalytic properties and stability of Akrilex C-carboxypeptidase A were studied and compared with the corresponding properties of the soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase A was practically identical to that for the soluble enzyme (pH 7.5–8.0). The apparent optimum temperature of the immobilized carboxypeptidase A was about 20°C higher than that of the soluble enzyme. With hippuryl- l-phenylalanine as substrate, K m app for the immobilized enzyme (1.65 mM) was somewhat higher than K m for the soluble enzyme (1.07 mM). The conformational stability of the enzyme was markedly enhanced by the strongly hydrophilic microenvironment. The immobilized carboxypeptidase A was used for the C-terminal amino acid analysis of peptides.

The direct electrochemistry of cryo-hydrogel immobilized myoglobin at a glassy carbon electrode

A cryo-hydrogel membrane (CHM) immobilized at a glassy carbon (GC) electrode is reported for the direct electron transfer of redox proteins. The most attractive characteristics of this CHM were its hydrophilic micro-environment for incorporated proteins to retain their activities, its high ability for protection against interference of denatured and adsorbed proteins at the electrode, its potential applications for various proteins or enzymes, as well as its high mechanical strength and thermal stability. A clear well developed and stable redox wave was obtained for commercially available horse heart myoglobin without further purification, giving a peak to peak separation ΔE p = 93 mV at 5 mV s −1 and the formal electrode potential E 0′ = −0.158 V (vs. Ag|Agcl). The formal heterogeneous electron transfer rate constant was calculated as k 0′ = 5.7 × 10 −4 cm s −1 at pH 6.5, showing rapid electron transfer was achieved. The pH controlled conformational equilibria, acid state η natural state η basic I state η basic II state, of myoglobin at the CHM GC electrode in the pH range 0–13.8 were also observed and are discussed in detail.

Ultraviolet resonance Raman spectra of pea intact, large, and small phytochromes: differences in molecular topography of the red- and far-red-absorbing forms.

Ultraviolet resonance Raman (UV RR) spectra excited at 244 nm were observed for pea intact, large, and small phytochromes at pH 7.8. Raman bands assignable to Trp residues dominated the UV RR spectra. The intensity ratios of Trp W7 doublet bands, I(1358)/I(1342), of all three phytochromes in the red light-absorbing form (Pr) were almost the same as that of an aqueous Trp solution, indicating that most of the six and four Trp residues in the 59-kDa chromophoric and the C-terminal 59-kDa nonchromophoric domains, respectively, reside in hydrophilic microenvironments in Pr. This ratio increased under red light illumination, where photoequilibria are attained between Pr and the far-red-absorbing form (Pfr) for intact and small phytochromes and among Pr, a bleached intermediate (Ibl), and Pfr for large phytochromes. The increase of the intensity ratio was most prominent for small phytochromes. These observations suggest that the microenvironments around some Trp residues become more hydrophobic due to conformational changes induced by phototransformation from Pr to Ibl and that the hydrophobicity increase occurs mainly in the chromophoric domain. Among the six Trp residues in the chromophoric domain, Trp365 and Trp567 are likely candidates for those involved in this hydrophobicity increase. The intensity distribution of the amide I band shows little beta-sheet in both Pr and Pfr of the intact, large, and small phytochromes and indicates that alpha-helices and nonregular structure are less populated in the chromophoric domain than in the N-terminal 6-kDa segment and the C-terminal nonchromophoric domain.

Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila.

Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD = 4 x 10(-9) M to 1 x 10(-8) M). Moreover, Scatchard transformations of the binding data show that there are about 5 x 10(5) binding sites per cell on the cell surface. Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdanLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl-beta-endorphin, is internalized preferentially into small vesicles. The differences may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein. Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).

Luminescence Probe Studies of Pyrene and Two Charged Derivatives in Nafion

The luminescence and absorption properties of pyrene, trimethylbyren-1-y1)ammonium chloride (tmpa) and sodium pyrene-1-sulfonate (sps) incorporated into the perfluorosulfonate membrane Nafion have been investigated. Addition of pb2+ induces a redistribution of pyrene molecules from the hydrophilic domains to more hydrophobic domains of the membrane and results in phosphorescence emission. tmpa is shown to bind strongly to the negatively charged sulfonate groups and does not exhibit phosphorescence nor is it displaced by pb2+. The fluorescence emission and absorption characteristics of sps incorporated in Nafion suggest a hydrophilic microenvironment, probably the water cluster region. The electrostatically unfavourable interaction, however, results in excimer formation even at low probe concentrations. Dynamic and steady-state fluorescence quenching measurements provide further insight into the nature of the different regions of Nafion.