Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme.

PURPOSE: The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products. METHODS: HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays. RESULTS: Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at -20°C. CONCLUSION: The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.

The effect of radiation-induced reactive oxygen species (ROS) on the structural and functional properties of yeast alcohol dehydrogenase (YADH).

PURPOSE: To determine the mechanism underlying oxidative modifications caused by radiation-induced reactive oxygen species (ROS) and elucidate their effect on the structure and function of yeast alcohol dehydrogenase (YADH), a zinc-containing protein. MATERIALS AND METHODS: YADH was exposed to water radiolysis products in an air atmosphere. YADH oxidation products were determined by spectrophotometric and spectrofluorimetric methods. The extent to which oxidative modifications affected enzyme activity was also studied. RESULTS: Water radiolysis products oxidized thiol groups leading to the release of zinc ions and the destruction of tryptophan and tyrosine residues. Those processes were accompanied by alterations in protein structure such as increased surface hydrophobicity, greater tryptophan accessibility to acrylamide, and changes in the secondary structure. Structural modifications were correlated with lower enzyme activity. CONCLUSION: During the process of functional and structural changes in YADH exposed to reactive oxygen species, a key part is the oxidation of cysteine residues attached to zinc and the release of zinc ions from the molecule. It may be assumed that ROS induce similar changes in many other zinc-containing proteins.

Inactivation of chosen dehydrogenases by the products of water radiolysis and secondary albumin and haemoglobin radicals.

PURPOSE: Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) by products of water radiolysis and by secondary radicals localized on haemoglobin (Hb) and human albumin (HSA) was studied. MATERIALS AND METHODS: Aqueous solutions of ADH, GAPDH and LDH were irradiated under air and under nitrous oxide (N2O) in the absence and in the presence of Hb or HSA. In order to determine the effectiveness of inactivation of the enzymes by radicals localized on Hb and HSA, the inactivation efficiency determined experimentally was compared with that calculated under assumption that only hydroxyl radicals are responsible for the enzyme inactivation. RESULTS: In the absence of other proteins, under air, GAPDH showed the highest radiation sensitivity, followed by ADH and LDH. The sequence was reverse under anaerobic atmosphere. Oxygen increased considerably the inactivation of GAPDH and ADH. Secondary albumin and haemoglobin radicals brought about considerable inactivation of GAPGH and ADH. Albumin radicals (HSA) generated under N2O inactivated GAPDH and ADH more effectively than haemoglobin radicals (Hb). Under air, however, inactivation of GAPDH and ADH by haemoglobin peroxyl radicals was higher than by albumin peroxyl radicals. LDH was resistant to inactivation by haemoglobin and albumin radicals, and peroxides of these proteins. CONCLUSIONS: In the light of these results and literature data, the observed differences in the effectiveness of inactivation of the dehydrogenases studied by secondary protein radicals depend on the amino acid residues present at the active site and in its close neighborhood and on the number of amino acid residues available on the protein surface.

The rate constants of the reaction of hydroxyl radicals (*OH) with alcohol dehydrogenase and Glyceraldehyde-3-phosphate dehydrogenase.

The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8 x 10(12) dm(3) mol(-1) s(-1), and 1.6 x 10(12) dm(3) mol(-1) s(-1), respectively.

De novo design and utilization of photolabile caged substrates as probes of hydrogen tunneling with horse liver alcohol dehydrogenase at sub-zero temperatures: a cautionary note.

In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.

[The possible role of the elements of protein secondary structure in adaptation to the action of ionizing radiation]. / O vozmozhnoi roli élementov vtorichnoi struktury belkov v adaptatsii k deistviiu ioniziruiushchei radiatsii.

Changes in the secondary structure of enzymes induced by gamma-rays 60Co at doses not exceeding one ionization per macromolecule were studied to elucidate a possible role of radiation-chemical processes in the evolution of proteins. The data on the comparative radioresistance of various types of secondary protein structures, alpha-helix, parallel and anti-parallel beta-structures, and beta-turn, were obtained by the method of circular dichroism. It was shown that beta-turns were resistant against radiation, alpha-helix was relatively stable, and beta-layer underwent significant changes. The importance of these structural types in the evolution of proteins is discussed. A special role of beta-turn as structural elements fixing the confirmation of macromolecules and therefore responsible for adaptation of the protein structure against a constant radiation background is proposed.

Potentiation of oxidative damage to proteins by ultraviolet-A and protection by antioxidants.

We have studied the damage of alcohol dehydrogenase (ADH) and glyceraldehyde 3-phosphate dehydrogenase (GAPD) induced by Fe++/EDTA + H2O2 in combination with UV-A (main output at 365 nm). Enzyme inactivation, formation of hydroxyl radicals (measured in the absence of enzymes), increase in protein carbonyls, oxidation of sulfhydryl (SH) groups, loss of native protein fluorescence, and enhanced protease degradation were used to determine protein damage. Hydroxyl radical production was greatly enhanced by the combination of UV-A with Fe++/EDTA + H2O2. The combined treatment increased protein carbonyls but decreased native protein fluorescence and SH groups. The combined treatment caused turbidity in GAPD but not in ADH, whereas trypsin susceptibility was increased more in ADH than in GAPD. These measurements of protein oxidation correlated well with enzyme activities. Glyceraldehyde 3-phosphate dehydrogenase and dithiothreitol were most protective against such damage, while hydroxyl radical and singlet oxygen scavengers were partially effective. Superoxide dismutase had no effect. Thus, UV-A potentiation of protein damage induced by FE++/EDTA + H2O2 appeared to involve hydroxyl radicals and perhaps singlet oxygen but not superoxide radicals. The damage to proteins induced by combination of UV-A with physiological oxidants, iron ions and H2O2 may be relevant to UV-A-induced skin and tissue damage.

Photophysical processes in the NADH-alcohol dehydrogenase complex.

Fluorescent analysis showed the high sensitivity of the NADH-alcohol dehydrogenase complex to UV irradiation. The complex reacts to UV illumination with "photoresponse," a rapid decrease in NADH fluorescence intensity to some stationary level. No such decrease was found in the NADH solution without the protein. The maximum rate of photoresponse was observed at pH 8.0, i.e, the biochemical optimum for the enzyme. It is suggested that the main reason for the photoresponse is desorption of NADH from the protein due to vibrational and conformational relaxation after photoexcitation. The data obtained point to the possibility of controlling "dark" enzymatic processes with light.

Synthesis and biological activity of N6-(N[(4-azido-3,5,6-trifluoro)-pyridin-2-yl]-2-aminoethyl)-adenosine 5'-monophosphate, a new AMP photoactivatable derivative. Covalent modification of horse liver alcohol dehydrogenase.

N6-(N-[(4-Azido-3,5,6-trifluoro)pyridin-2-yl]-2-aminoethyl)- adenosine 5'-monophosphate has been synthesized and evidence presented for its structural assignment by ultraviolet and 19F-NMR spectroscopies. Its photolysis was shown to occur within 5 min. This AMP derivative behaves as a competitive inhibitor of NAD+ in horse-liver-alcohol-dehydrogenase-promoted oxidation of ethanol, with a Ki (0.95 mM) comparable to the Ki of AMP (1.9 mM). Moreover it is an activator of the enzyme when nicotinamide ribose is used as the oxidation cofactor. This activation is as good as that promoted by AMP or by the well known 8-azido-AMP. Upon photolysis of this new derivative in the presence of horse liver alcohol dehydrogenase, a covalent enzyme--analogue complex was isolated and assayed as a catalyst in the oxidation of ethanol using nicotinamide ribose as the cofactor. The reaction took place without complementation of AMP, indicating clearly that the AMP analogue is mainly covalently bound in the AMP-binding site, and that the linkage formed between the enzyme and the azido derivative has not dramatically altered the active site of the enzyme. A similar experiment with 8-azido-AMP produced a completely inactive complex.

Zinc release from irradiated yeast alcohol dehydrogenase.

The release of Zn2+ from gamma-irradiated yeast alcohol dehydrogenase has been measured using atomic absorption spectrometry. Radiolysis is accompanied by release of Zn2+ at a rate which is dependent on the nature of the free radicals available for reaction. Hydroxyl radicals and hydrogen atoms readily cause zinc release with G values of 0.13 and 0.11 (/100 eV) respectively, whereas hydrated electrons are considered not to contribute to the demetallization process. The radiolytically generated radical anions I2-., (SCN)2-. and Br2-. enhance the rate of zinc release. Evidence is presented that the enzyme is demetallized as a result of free radical reactions at cysteine and histidine residues.

Yeast and horse liver alcohol dehydrogenases: potential problems in target size analysis and evidence for a monomer active unit.

Yeast and horse alcohol dehydrogenases are commonly used as standards for radiation inactivation analysis of proteins, usually assuming that the minimal functional unit corresponds to the physical size in solution, a tetramer (Mr = 148,000) and a dimer (Mr = 80,000), respectively. Results described in this paper demonstrate that molecular weight overestimates may be obtained for the yeast protein as a result of its unusual sensitivity to secondary radiation products. Irradiation in the presence of sulfhydryl reagents results in a smaller functional size estimate (67,000 +/- 3000) than that obtained in their absence (128,000 +/- 5000), indicating that some sulfhydryl groups in the enzyme may be particularly susceptible to attack by radiolytic species. Analysis of the horse liver enzyme reveals that although it has structural and functional similarities to the yeast protein, it is not as prone to secondary radiation damage and gives a minimal functional size estimate (33,000 +/- 1000) that most closely corresponds to a monomer. Quantitation of disappearance of the protein from a sodium dodecyl sulfate gel as a function of radiation dose also gives a target size (48,000 +/- 3000) in reasonable agreement with the monomer molecular weight. These results indicate that the individual subunits of horse liver alcohol dehydrogenase have independent catalytic capacity and imply that the same may be true for the yeast enzyme.