Summ ary2)The quaternary ammonium salt methyl-trioctylammonium chloride enables the transfer of a-chymotrypsin, trypsin, pepsin and glucagone from water to cyclohexane. Reversed micelles, whose polar core solubilizes both protein and water, are probably formed in the apolar phase. The influence of various parameters on the phase transfer (concentration, pH, solvent, temperature, etc.) has been investigated.Absorption, fluorescence and circular dichroism studies of the biopolymers in the cyclohexane system have been carried out. For trypsin and chymotrypsin, the CD. signal in the 200 nm region is very similar in water and in Icyclohexane, which suggests that the polypeptide folding is not substantially different in the two phases. The fluorescence quantum yield is always much larger in the cyclohexane phase than in water. The longer wavelength region of the UV. absorption spectrum is slightly red-shifted relative to water, and a band at 225 nm, probably arising from the aromatic chromophore, is apparent in the organic phase. Reasons for these spectral perturbations are discussed. The enzymes transferred from water into cyclohexane phases can be continuously retransferred into a second water phase. The possible relevance of this 'double transfer' as a model for the vectorial transport of biopolymers or a separation technique is discussed.

The fluorescence properties of horse-liver alcohol dehydrogenase were investigated with the aim of separating the contribution of Trp-15 (which is close to the protein surface) from that of Trp-314 (buried in the interior of the protein). Quenching of the protein fluorescence by iodide involves, to a larger degree, the longer wavelength region of the protein emission spectrum and is interpreted to involve only , , at 340 nm and a quantum yield of 0.19). It is shown that quenching by NAD and NADH involves both types of tryptophan, but the 'blue' one to a larger extent. In the case of NADH, radiationless energy transfer between enzyme and reduced nicotinamide ring accounts for less than one half of the total protein fluorescence quenching. Energy transfer between the tryptophan and adenine rings is also possible, but it cannot account for the rest of the protein quenching. Thus, it is suggested that protein conformational changes, following NADH binding, are the cause of part of the fluorescence quenching. The extent to which quenching by NAD can be ascribed to radiationless energy transfer processes is also calculated. It is shown that despite the small spectral overlapping between coenzyme absorption and protein emission, the energy transfer contribution cannot be neglected. However, it is very likely that also in this case a sizeable part of the protein fluorescence quenching comes from protein conformational changes following coenzyme binding. The possible nature of these conformational changes is discussed, taking into account recent X-ray data of enzyme-coenzyme complexes.Horse-liver alcohol dehydrogenase has four tryptophan residues, two per subunit of molecular weight 40000 [l]. Recent X-ray data locate them spatially: Trp-15 is close to the surface and probably exposed to water, while Trp-314 is buried inside the protein, close to the interaction domain of the two subunits property has been used in a number of ways in order to study the interaction between the apoenzyme and coenzyme [4-81. The quenching produced by binding of NADH is usually attributed to energy transfer [5,8] whereas the quenching upon binding of NAD, analogous to the cases of octopine dehydrogenase [9] and yeast alcohol dehydrogenase [I 01, has been ascribed to a protein conformational change [7]. The present work analyzes the overall protein fluorescence in terms of the contribution of the two types of tryptophan residues. It will be shown that it

SynopsisThe fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (Am=) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-TrpGly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly),-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of A, ., , and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the Amax of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.

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