General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.

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Time-resolved fluorescence and fluorescence anisotropy experiments were conducted on rhodamine B and octadecylrhodamine B incorporated into aqueous Triton X-100 micelles and in sodium bis(2-ethylhexyl) sulfosuccinate entrapped water or glycerol in a hydrocarbon solvent (heptane or dodecane). The time-resolved fluorescence behavior of the dye molecules in the micellar media was compared with that of the dye in homogeneous solution, from which a qualitative estimate of the polarity of the probe environment in the micelles could be inferred. The anisotropy decay of the fluorescent probes was analyzed with a biexponential decay model yielding correlation times characteristic for overall and internal micellar motion. The overall micellar rotation could be clearly distinguished from the faster internal motion in small water droplets and in glycerol droplets in heptane, for which there is good agreement between calculated and observed micellar rotation times. The hydrodynamic radii of glycerol droplets in dodecane medium are larger than the corresponding radii of droplets in heptane.

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH,) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH, decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH, and 1.099 ns for p-terphenyl (20°C), whereas the AcTrpNHz decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency.The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 "C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for A,, = 295 nm and 36 ns for ,Iex = 300 nm at 20°C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, whch give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.Time-resolved fluorescence measurements of intrinsic probes in proteins can be a powerful tool to obtain information about the molecular structure and dynamic processes taking place on the fluorescence time scale, like rotation of the whole molecule or segments of it and interactions with other molecules, present in the solution [l, 21. One class of intrinsic probes is formed by the tryptophan residues in proteins [3].Interpretation of tryptophan fluorescence decay is a complex matter. In many cases, multiple exponential decays are found for proteins containing a single tryptophan [4]. This anomalous behaviour has been explained by the existence of three different a -/ 3 rotamers of tryptophan. According to the so-called 'modified conformer model' (MCM) [5 -71 intramolecular charge transfer from the excited indole ring to electrophilic substituents on the 3-ethyl chain can occur. In each of the three a-p rotamers the di...

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Znporphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrimethylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces.Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (w, = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved.Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions.In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.Surfactant assemblies in organic media, called reversed micelles or, more general, water-in-oil microemulsions, have been investigated extensively over the past years. Many studies have been carried out towards the elucidation of structure and dynamics of reversed micelles [I -41 but also on the (bio)chemical and (bio)technological applications of these systems [2, 3, 5-91. In our laboratory an important line of research consists of the incorporation of proteins in reversed micelles in order to perform bioconversions of apolar compounds or to isolate proteins [lo -121. At present, the effects of protein solubilization on both protein and micelle structure and dynamics are not very clear. Because reversed micellar solutions are optically transparent, spectroscopic and ultracentrifugation techniques can be applied to study these effects. Several systems have been the subject of this kind of investigation, some containing cytoplasmatic, others containing membrane proteins [5, 7-91. Horse heart cytochrome c is a protein which in vivo is strongly associated via electrostatic interactions with the inner Correspo...