Proteins radiolysis

Adams, G.E. and Redpath, J.L. (1974). Selective free-radical reactions with proteins and enzymes pulse radiolysis and inactivation studies on papain. Int. J. Badiat. Biol. 25, 129-136. 

Table 2 contains the computational results of structural Pc-parameters of free radicals by the equation (8). The calculations are made for those radicals forming protein and aminoacid molecules (CH, CH2, CH3, NH2, etc), as well as for free radicals being formed during radiolysis and dissociation of water molecules (H, OH, H30, H02). 

Flash photolysis and pulse radiolysis techniques have been developed to study Fe Ru ET in Ru-modified proteins [21,26,27]. A method that allows study of electron transfer from a surfaee ajRu(IIIXhistidine) to a protein redox center is outlined in the Scheme [21]. The ET reaction is initiated by photogenerated... 

Rate constants for azurin Ru(NH3)5-modified at His83 have been determined by two methods in which the fully oxidized Ru(III)Cu(II) protein is reduced to Ru(II)Cu(II) by (a) flash photolysis using [Ru(bipy)3] as re-ductant [128], and (b) pulse radiolysis with CO2 as reductant [50]. Intramolecular rate constants of 1.9 + 1.4s (22°C) and 2.5+0.8 s (four runs at 17 °C), respectively are in reasonably satisfactory agreement. Both studies were in 0.10 M phosphate, but solutions for pulse radiolysis also have in addition 0.10 M sodium formate present. Ionic strengths were therefore 0.22 and 0.32 M (the value of 0.31 M in Refs. [50] and [129] is closer to and should be adjusted to the latter value in Ref. [130] an ionic strength of 0.32 M applies to the and O2... 

On pulse radiolysis using CO f (—2.0 V) to reduee PCu(II)Ru(III) (pH 7, 20 °C) the behavior observed is in both cases very similar [50]. The concentration of C02 was adjusted so that there was <20% reduction of modified protein. As far as can be ascertained, reduction efficiencies in the first stage are about the same, with reaction partitioned between the Cu(II) (72%) and the... 

The intramolecular electron transfer kg, subsequent to the rapid reduction, must occur because the Ru(III)-Fe(II) pairing is the stable one. It is easily monitored using absorbance changes which occur with reduction at the Fe(III) heme center. Both laser-produced Ru(bpy)3 and radicals such as CO (from pulse radiolysis (Prob. 15)) are very effective one-electron reductants for this task (Sec. 3.5).In another approach," the Fe in a heme protein is replaced by Zn. The resultant Zn porphyrin (ZnP) can be electronically excited to a triplet state, ZnP which is relatively long-lived (x = 15 ms) and is a good reducing agent E° = —0.62 V). Its decay via the usual pathways (compare (1.32)) is accelerated by electron transfer to another metal (natural or artificial) site in the protein e. g.. 

The kinetics of myoglobin oxidation and reduction have been studied by a variety of experimental techniques that include stopped-flow kinetics, pulse radiolysis, and flash photolysis. In considering this work, attention is directed first at studies of the wild-type protein and then at experiments involving variants of Mb. 

Pulse radiolysis has also been used to study the reduction of various Mb derivatives by hydrated electrons 151-154). With this technique, it was possible to study reduction of ligand-bound forms of metMb at cryogenic temperature and thereby identify reduced, ligand-bound forms of the protein 152) and to reduce oxyMb to produce ferryl (Fe(IV)=0)... 

Racemizations in the crystalline state have a long history. It is known that L-a-amino acids slowly racemize in the solid state [62]. As this also happens in solid proteins the implications are manifold, not only in pure chemistry but also in biochemistry, nutrition, food technology, and geology. Therefore, techniques have been developed to determine the dl ratio of amino acids down to 0.1% and inversion rate constants have been determined under acid hydrolysis conditions [63]. One could think of very slow deamination and readdition of the amine or an enolization mechanism. However, such reactions can also be induced by photolysis or radiolysis from natural sources [64]. 

Pecht, I., Goldberg, M. Electron transfer pathways to and within redox proteins Pulse radiolysis studies. In Fast Processes in Radiation Chemistry and Biology (Adams, G. E., Fielden, E. M., Michael, B. D., eds.), Wiley, Chichester-New York-Toronto-Sydney, 1975, pp. 277-284... 

Electron transfer between the iron centre in cytochrome c and a transition metal bound at the periphery of the protein has been studied in cases where Cu" 677 and the Rum(NH3)5 group678 are bound to His-33. In the latter case, the Ruu-cytochrome c(III) form can be generated by pulse radiolysis. The intramolecular transfer of an electron between Ru" and Fe111 can be measured. This presents a useful model for electron transfer from the iron via the heme group and the protein. 

Re-examination of the radiolysis of aqueous solutions of alanine (absence of oxygen) shows that electrons react rapidly with the cationic form, less rapidly with the zwitterion, and much less rapidly with the anionic form. These conclusions have been confirmed by pulse radiolysis. Rate constants for amino acids, peptides, proteins, and numerous other substances have been obtained. Critical evaluation of these and correlation with molecular properties is now well under way. In living systems the reactions of the hydrated electron vary with the part of the cell concerned, with the developmental stage of the cell, and possibly with the nature of any experimentally added substances. 

To acquire further insight into the mechanism of the formation of these compounds, particularly of the protein-derived components, the radiolysis of several amino acids was studied. In all the amino acid studies, solutions or slurries of the acid were used in amounts corresponding to the... 

Interaction of the solute with radicals from the water is the first of a sequence of reactions which finally leads to stable products. Kinetic studies of the type cited give valuable information about the primary radical species and their relative reaction rates with molecules of different types. When sufficient data have been accumulated, it should be possible to predict the course of radiolysis in complex molecules. From the nature and yields of the products and by observing the effects on them of various factors such as concentration, pH, 02, and specific radical scavengers, it is often possible to speculate about the mechanisms by which products are formed. More often than not, this is a difficult problem because the products, even from relatively simple compounds, prove to be complex. Furthermore, it is often possible to produce more than one mechanism to fit the experimental data. The proteins are particularly difficult because of their complex structures. They contain approximately 20 different amino acids with an average of more than three carbon atoms in the side chains, which vary considerably in their structure hence, the possible number of products is large. For this reason, model compounds such as peptides and polyamino acids have been studied because they contain the peptide linkage but are free from the complications which arise from the diversity of the amino acid residues in a protein. A further practical difficulty which applies to chem-... 

Dizdaroglu M, Schulte-Frohlinde D, von Sonntag C (1977) y-Radiolysis of DNA in oxygenated aqueous solution. Structure of an alkali-labile site. Z Naturforsch 32c 1021—1022 Dizdaroglu M, Gajewski E, Reddy P, Margolis SA (1989) Structure of a hydroxyl radical induced DNA-protein cross-link involving thymine and tyrosine in nucleohistone. Biochemistry 28 3625-3628... 

Begusova M, Gillard N, Sy D, Castaing B, Charlier M, Spotheim-Mauizot M (2005) Radiolysis of DNA-protein complexes. Radiat Phys Chem 72 265-270 Bellon S, Ravanat J-L, Gasparutto D, Cadet J (2002) Cross-linked thymine-purine base tandem lesions synthesis, characterization, and measurement in y-irradiated isolated DNA. Chem Res Toxicol 15 598-606... 

Schuessler FI, Schmerler-Dremel G, Danzer J.Jung-Korner E (1992) Ethanol radical-induced protein-DNA crosslinking. A radiolysis study. Int J Radiat Biol 62 517-526 Schuessler FI, Distel L, Sieber R (1997) Radiolysis of DNA in the presence of a protein studied by HPL-gel chromatography. Int J Radiat Biol 71 543-553... 

All amino-acid residues of proteins are potential targets for attack by reactive oxygen species (ROS) produced in the radiolysis of water however, in only a few cases have the oxidation products been fully characterized. Moreover, under most physiological conditions, cysteine, methionine, arginine, lysine, proline, histidine, and the aromatic amino acids are primary targets for ROS-mediated oxidation. 

The reaction Fe ccp/Fe cytc + Fe ccp/Fe cytc proceeds with AE s 0.4V. The reaction h j been moj ored both by pulse radiolysis, and by simple mixing of Fe ccp + Fe1 cytc, with equivalent results k 0.25 0.07 s (figure 10) It is interesting that a dependence of rate on the primary structure of the protein is observed (at constant AG) for horse cy1j.c/ccp(yeast) k = 0.25 s but for yeast. cytc/(yeast) ccp k = 4 s 1 and for tuna cytc/yeast ccp k s 0.1 s, even though the general three dimensional structures are essentially identical for horse, tuna and yeast cytochromes c. These determinations disprove an earlier suggestion based on modulated excitation spectroscopy, that k - 10 s. Clearly the rate is slow,... 

SCHEME 4.1 Schematics of radiolysis and reducing species. As a result of ionization of the water molecule, hydroxyl radicals and hydrated electrons are formed. The final radiolytic yield depends on the secondary reactions in spurs and on the presence of other compounds. See Refs 25,26,190, and 191 for the detailed discussion and references. Solvated electrons are mobile enough to escape spurs and to react with the heme protein complexes even at 77K. All other reactive products of radiolysis are immobilized in the solid solvent matrix, or trapped by radical quenchers. 

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