The 60 Co g-Radiolysis of Aqueous Papain Solutions: Repair and Protection by Cysteine

Gaucher, G. M.; Mainman, Barbara L.; Thompson, Gail P.; Armstrong, David A.

doi:10.2307/3573427

Cited by 21 publications

(13 citation statements)

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“…Furthermore, as shown in increasing dose. This can be attributed to the destruction of one or more of the three disulfide I bridges ofpapain, evidence for which has already been obtained in the 60Co y-radiolysis of the enzyme (7). The fact that the absorption at 3 10 nm changes less with increasing dose suggests that much of the intensity at the lower wavelengths is due to different species.…”

Section: Reactions Of E Lmentioning

confidence: 80%

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Pulse Radiolysis of Aqueous Papain

Clement¹,

Armstrong²,

Klassen³

et al. 1972

Can. J. Chem.

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The pulse radiolysis of the proteolytic enzyme papain has been studied in nitrogen-purged and N2O-purged aqueous solutions at pH 6.4. The rate constant for reaction of OH with papain is 4.7 × 1010 M−1 s−1. The initial spectrum of the intermediate formed by this radical is consistent with a major fraction of attack at tyrosine residues with smaller proportions reacting at tryptophan and other sites. Subsequent spectral changes are also consistent with the preferential attack of OH on tyrosine. The overall rate constant for reaction of [Formula: see text] with papain is 4.1 × 1010 M−1 s−1 and the spectrum of the electron adduct closely resembles that of the disulfide anion [Formula: see text] However, there is evidence that not all reactions of [Formula: see text] with papain lead to the formation of this species. The results are discussed in the light of deactivation yields for Co60γ radiolysis measured by biochemical techniques.

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Section: Reactions Of E Lmentioning

confidence: 80%

“…In an earlier paper (7) we reported the results of a 60Co y-radiolysis of papain. The enzyme exhibited a high sensitivity to inactivation by [Traduit par le journal] e,, a large part of which could be repaired by postirradiation treatment with cysteine.…”

Section: Introductionmentioning

confidence: 99%

Pulse Radiolysis of Aqueous Papain

Clement¹,

Armstrong²,

Klassen³

et al. 1972

Can. J. Chem.

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“…cysteine. [3] The protection obtained when cysteine is present during irradiation in a [cysteine]:[papain] ratio of 10:l has been reported previously (8). However at low [cysteine]:[papain] ratios, cysteine was found to increase the amount of repairable damage (9).…”

Section: Introductionmentioning

confidence: 89%

“…Re/~(iir 01' P(i/~(iitz f i~( i c t i~~~i / c~/ by Hj~drogen Perax-ick~ (i) Reactions 4 and 5 ~iormally proceed to complctioli at 3 0°C in thc post-inoctivatioli repair assay due to the 30 min treatment with 21 1000-fold excess of cysteine (8). This repair 111ay 21lso occur to sonie cxtcnt at 25 "C with lower cystcine concciitrations.…”

Section: Resultsmentioning

confidence: 99%

“…After tlie repair or nonrepair treatment the enzyme solutions were assayed for enzyme activity sing N-CBZglycine-11-nitrophenyl ester as s~~b s t r a t e (8). Losses in activity were calculated from the tlilference between the activity of a n unirraciiatetl but otherwise identically treated control and the activity of the irradiated solution.…”

Section: Procerlr~resmentioning

confidence: 99%

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Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H₂O₂ and OH radicals

Lin¹,

Gaucher²,

Lal³

1976

Can. J. Chem.

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Chenl. 54, 242 (1976).The inactivation of the proteolytic enzyme papain by hydrogen peroxide produces a sulfenic acid by oxidation of the essential SH of cysteine 25 at the enzyme active site:The kinetics of repair of this entity by cysteine were consistent with the two reactions:[4 1 papainCys25SOH + CysSH 4 papainCys2sSSCys + H20 151 papainCys2sSSCys + CysSH -r papainCys2sSH + CysSSCys Reaction 4 was the faster with k4 2 800 M-1 s-1, and ks = 11.3 T 0.5 M-1 s-1. A computer program was developed to evaluate the contributions of peroxide-inactivation and cysteinerepair when they occur sinlultaneously in NzO-saturated solutions in the absence of catalase. The yields predicted by this program agreed well with the inactivation caused by peroxide in irradiated systelns.The effect of cysteine 011 the inactivation of papain by O H radicals produced by radiolysis of NlO-saturated solutions containing catalase was also investigated. Protection against permanent inactivation was n~uch more efficient than expected on the basis of a simple competition between cysteine and papain for O H radicals, but there was a marked increase in the yield of repairable damage which was not due to hydrogen peroxide. These observations can be qualitatively accounted for by the reactions:The same rate constant was obtained for the repair of PapainCys2jSSCys from this source as from the peroxide inactivation and treatment with cysteine. However, there was also evidence for additional cysteine-sensitized production of mixed disulfide and this probably occurs t h r o~~g h reactions of CysS. radicals: Chem. 54, 242 (1976). L'inactivation de I'enzyme protkolytique de la papaine par le peroxyde d'hydrogkne conduit h la formation d '~~n acide sulfknique par oxydation du SH essentiel de la cystiine 25 au niveau du site actif de I'enzyme: O n a aussi etudii I'effet de la cystiine sur I'inactivatio~l de la papaine par les r a d i c a~~x O H p r o d~~i t s par la radiolyse de solutions saturies de N?O contenant la catalase. La protection contre une inactivation permanente est beaucoup plus eficace qu'attendue sur la base d'une simple conlpititio~l entre la cysteine et la papaine pour les radicaux O H , ~n a i s il y avait une augmentation marquee d a m le rendenlent de dornmage reparable qui n'itait pas dfi au peroxyde d'liydrogene. O n peut tenir compte qualitativenle~lt de ces observatiolls par les riactions:p a p a i n e C y~~~S .-+ (donne des pertes de S H non-riparables)On a obtenu les mimes constantes de vitesse pour la riparation de la papaineCys25SSCys B partir de cette source et aussi B partir d'une inactivation par le peroxyde et le traitenlent avec la cysteine. Toutefois, il y a aussi des indiquations pour la production se~lsibilisie par la cysteine du disulfure rnixte et ceci se produit vraisemblablenle~lt par des reactions des radicaux CysS.:

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The Use of Free Radical Probes in the Study of Mechanisms of Enzyme Inactivation

Adams

Redpath

Bisby

et al. 1972

Israel Journal of Chemistry

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Some inorganic radical‐anions, which have very strong absorption spectra, have some oxidative reaction specificity with amino acids. Combined pulse radiolysis and inactivation studies on the reactions of these radicals with several enzymes lead to identification of amino acid residues essential to enzymic activity. Data are reviewed for the enzymes, lysozyme, ribonuclease, trypsin, α‐chymotrypsin, papain and carboxypeptidase. The reducing radical, CO2−, is more selective than the hydrated electron in its reactions with enzymes and proteins. Results from pulse radiolysis studies in electron‐transfer and other reactions involving CO2− are compared with results from inactivation studies with lysozyme and ribonuclease.

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The 60 Co g-Radiolysis of Aqueous Papain Solutions: Repair and Protection by Cysteine

Cited by 21 publications

References 28 publications

Pulse Radiolysis of Aqueous Papain

Pulse Radiolysis of Aqueous Papain

Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H₂O₂ and OH radicals

The Use of Free Radical Probes in the Study of Mechanisms of Enzyme Inactivation

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The 60 Co g-Radiolysis of Aqueous Papain Solutions: Repair and Protection by Cysteine

Cited by 21 publications

References 28 publications

Pulse Radiolysis of Aqueous Papain

Pulse Radiolysis of Aqueous Papain

Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H2O2 and OH radicals

The Use of Free Radical Probes in the Study of Mechanisms of Enzyme Inactivation

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Cysteine-sensitised formation and repair of mixed disulfide in the oxidation of papain by H₂O₂ and OH radicals