L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.

Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This article describes a general method for detecting pegylated proteins directly after SDS-PAGE. The proteins to which polyethylene glycol (PEG) molecules are attached are stained with a barium iodide solution. The staining is based on the formation of a barium iodide complex with PEG. The described method combines a specific staining of PEG molecules with the high resolution of the SDS-PAGE method. It is shown that pegylated protein is detectable on SDS-PAGE as well as on IEF at concentrations that are not detectable by Coomassie protein staining. This paper also describes the determination of the molecular weight of pegylated hirudin by calibrating SDS-PAGE with polyethylene glycol of different molecular weight. Under the conditions used, PEG showed linear mobility during electrophoresis. However, the use of nonpegylated proteins as standards resulted in incorrect molecular weight values due to the lower mobility of the pegylated protein during electrophoresis. The method described might reflect a general method for determining molecular weight of pegylated proteins.

Bioluminescence emission of bacterial luciferase with 1-deaza-FMN. Evidence for the noninvolvement of N(1)-protonated flavin species as emitters.

The reaction of reduced 1-d-FMN with oxygen and decanal results in bioluminescence with kinetic and spectral properties similar to those of the reaction with FMNH2, even though the spectral (absorbance, fluorescence) and chemical properties of the oxidized forms differ greatly. This emission, which is about 10-15% as efficient as with FMNH2, is postulated to involve the intermediacy of the corresponding 4a-hydroperoxide, the fluorescence of which occurred transiently. The N(1) protonated species had been proposed as the emitter in the reaction with FMNH2, but the 1-deaza analog cannot be protonated at the corresponding position, thus excluding this possibility.

Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction.

An intermediate identifiable as the emitter in bacterial bioluminescence has been demonstrated. The reaction was carried out at 1 degrees C by mixing purified luciferase-bound FMN 4a-hydroperoxide with long-chain aldehyde (decanal). Simultaneous kinetic measurements of bioluminescence and absorbance showed that the decay of light emission occurred more rapidly than the appearance of the stable product, oxidized FMN, indicating the formation of a transient intermediate species subsequent to light emission. The same species was found in reaction mixtures examined immediately after light emission was completed. It has a relatively short half-life (7 min at 9 degrees C); the chromophore is postulated to be the luciferase-bound flavin 4a-hydroxide and to decay to the stable product, FMN, by losing water. Both its absorption spectrum (lambda(max), 360 nm) and its fluorescence emission (lambda(max), 490 nm) are consistent with the hypothesis that this is the ground state of the primary emitter, the bioluminescent species produced in the reaction.

Bioluminescence emission from the reaction of luciferase-flavin mononucleotide radical with O2-..

The blue neutral luciferase flavin radical has been shown not to be in a catalytically significant equilibrium with species leading to emission of light [Kurfürst, M., Ghisla, S., Presswood, R., & Hastings, J. W. (1982) Eur. J. Biochem. 123, 355-361]. It is shown here that this radical can nevertheless react with O2-. to form a species that is competent in light emission. From its properties, the species formed is deduced to be luciferase-FMNH 4a-hydroperoxide, a key intermediate in the normal luciferase reaction. Although it is concluded that this intermediate can undergo a reversible homolytic dissociation to yield free superoxide and the corresponding luciferase radical, the slowness of these steps precludes a catalytic significance for these pathways in the normal bioluminescent reaction.

Structure and catalytic inactivity of the bacterial luciferase neutral flavin radical.

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase-FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 approximately 20 h at 0 degree C in air; the Arrhenius delta H not equal to decay being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 degrees C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction.