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
Reduced 3-thionicotinamide -adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 The interaction between sNADH and octopine dehydrogenase was investigated by ultraviolet-difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine, as follows. (a) There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40-nm blue shift of the emission maximum. (b) The protein fluorescence is quenched almost completely. (c) The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring in the free form. These features appear to be unique to the octopine. sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. It was found that sNAD neither binds nor acts kinetically as a coenzyme. sN4DH exhibits relatively good binding, with K, and Ki values close to those of the natural coenzyme, but the turnover number is 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH dissociation constants were determined as a function of temperature, and appear to be practically temperature-independent in the range 10 -40 "C. It seems thus, in agreement with previous studies, that the interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity, and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed. 0.5) xOctopine dehydrogenase from Pectm maximus L., which catalyzes the reversible oxidation of D-octopine by NAD to give L-arginine plus pyruvate, was first isolated by van Thoai and collaborators [I]. The enzyme has a B stereospecificity towards NADH and has a fairly restricted specificity towards substrates and coenzymes [l -41. Detailed studies have been made of the spectroscopic properties of various complexes [5 -71, the binding of coenzymes in the presence of various ligands [8-lo], and of the kinetic mechanism [l I, 121. One of the most interesting properties of octopine dehydrogenase is the temperature dependence of sev-
The interaction between horse liver alcohol dehydrogenase and the oxidized and reduced forms of the 3-thionicotinamide -adenine dinucleotide coenzyme analogues (sNAD and sNADH) has been investigated by ultraviolet absorption, fluorescence and circular dichroism. The fluorescence of sNADH is enhanced when bound to the enzyme, and the protein fluorescence is quenched by both sNADH (60-65 %) and sNAD (65 %). The possible origin of the larger quenching produced by sNAD with respect to that of NAD is discussed. Coenzyme dissociation constants have been determined by monitoring the quenching of the protein fluorescence, and some kinetic consequences of these dissociation constants are discussed. The dichroic properties of various enzyme complexes have been investigated, and are discussed in terms of conformational changes and environmental changes during coenzyme binding. The conformation of sNAD bound to the enzyme in the presence of trifluoroethanol and the conformation of sNADH bound to the enzyme in the presence of isobutyramide have been analyzed in particular detail. Also the circular dichroic spectrum of the apoenzyme is discussed in terms of contributions of the aromatic amino acid residues in the highly resolved 240-310-nm region and in terms of helix content in the 220-nm region.
The hypothetical release of 3,3'-dichlorobenzidine (DCB) from two insoluble azo pigments and from a soluble azo dye was investigated in female Wistar rats for a 4 week treatment with 0.2% (w/w) Colour Index Pigment 13 (PY13) or 0.2% (w/w) Colour Index Pigment Yellow 17 (PY17) in the diet or 0.06% (w/v) Colour Index Direct Red 46 (DR46) in the drinking water. Steady-state DCB-hemoglobin adduct levels were determined by GC/MS with negative chemical ionization as well as DCB-DNA adduct levels in the liver by (32)P-postlabelling and compared with the respective adduct levels obtained in animals after treatment for 4 weeks with 0.00024, 0.0012 or 0.006% (w/v) DCB in the drinking water. A dose-proportional increase in adduct levels from 8.1 ng/g hemoglobin and 2.6 ng/g DNA (relative adduct level, RAL, 3.3x10(-9)) to 160 ng/g hemoglobin and 45.4 ng/g DNA (RAL 56.1x10(-9)) was observed in the DCB-treated rats. In rats treated with DR46 total adduct levels of 17.7 ng/g hemoglobin and 5.2 ng/g DNA (RAL 6.4x10(-9))were determined. No hemoglobin of DNA adducts were found in rats treated with PY17 in the diet, at a limit of detection of 0.1 ng/g hemoglobin and 0.08 ng/g DNA (RAL 0.1x10(-9)). In animals treated with PY13 in the diet no adducts or only minimal amounts slightly above the limit of detection could be identified. Taking into consideration that PY13 was contaminated with 0.02% of the respective soluble monoazo compound, it is concluded that the small amounts of DCB detected have been released from the contaminating soluble monoazo compound and not from insoluble PY13. The results of the present study demonstrate the lack of bioavailability of DCB from the diarylide azo pigments PY17 and PY13.
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