Biomedical subjects

K J Stevenson

Publications and source records attributed to K J Stevenson.

At least 37 records · Page 2Linked to original sources

PubMed1989

Conformational analysis of thioredoxin using organoarsenical reagents as probes. A time-resolved fluorescence anisotropy and size exclusion chromatography study.

Reduced thioredoxin was subjected to chemical modification studies employing organoarsenical reagents specific for "spatially close" thiols. Modification was monitored by the loss in the free thiol content, by the percent incorporation of radiolabelled organoarsenical reagents, and by observing the changes in the amounts of the various thioredoxins by size exclusion chromatography. The rate of modification depends upon the polarity, rigidity, and size of the reagents. Small nonpolar organoarsenical reagents readily modified reduced thioredoxin, whereas polar and large reagents do not. Modifications resulted in the formation of stable 15-membered cyclodithioarsenite ring structures with no apparent changes in the secondary structure of the protein. Modification was reversed by the extrusion of the arsenical moiety by addition of 2,3-dimercaptopropanol. We have further characterized the oxidized, reduced, and modified thioredoxins by size exclusion chromatography and fluorescence anisotropy decay measurements. Both techniques show an increase in the hydrated volume of the protein upon reduction. Upon modification, the hydrodynamic volume of the protein further swells. Fluorescence anisotropy decay reveals that with modification there is loosening of the protein so that a "domain" containing the fluorophores can relax independently of the whole protein structure.

Alkylation↗

PubMed1988

Structural comparison between oxidized and reduced Escherichia coli thioredoxin. Proton NMR and CD studies.

Escherichia coli thioredoxin (Mr 11,700) usually functions as a hydrogen carrier protein that undergoes reversible oxidation/reduction reactions of its active-site disulfide linkage. By use of a number of assigned and identified resonances in one- and two-dimensional 1H NMR spectra, the two forms of the protein have been compared. Only groups that are relatively close to the active-site Cys-32, Cys-35 linkage such as Trp-28, Trp-31, Phe-27, Ala-29, and Val-25 undergo substantial changes in their 1H NMR chemical shift upon reduction. Various residues that are further removed from the active site, like Tyr-49, Tyr-70, His-6, Phe-12, Phe-81, and Phe-102, appear to be little affected (less than 0.02 ppm) by the reduction, suggesting that the rest of the protein structure is not much affected. Thus, the structural changes that occur upon reduction appear to be localized to the disulfide-containing turn and the central strand of the twisted beta-sheet that directly leads to this turn. Notwithstanding the apparent similarity in the secondary and tertiary structures of the oxidized and reduced forms of the protein, the thermal stability of the protein decreases by 10 degrees C upon the reduction of the single disulfide. This was found by both 1H NMR and near- and far-ultraviolet circular dichroism studies. Oxidized thioredoxin was also more resistant to alkaline denaturation. Furthermore, the exchange rate of the relatively stable slow-exchanging backbone amide protons that are part of the core of the twisted five-stranded beta-sheet of thioredoxin increases substantially after reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1988

Human plasma lecithin-cholesterol acyltransferase. The vicinal nature of cysteine 31 and cysteine 184 in the catalytic site.

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. We proposed a covalent catalytic mechanism of action for LCAT (Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7034) in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols we have probed the geometry of the catalytic site. p-Aminophenylarsendichloride noncompetitively inactivates cholesterol esterification (Ki = 0.23 mM) by LCAT via alkylation of both catalytic cysteine residues. This reagent does not significantly inactivate lecithin cleavage by LCAT. Full enzyme activity is restored by treatment with 2,3-dimercapto-1-propanesulfonic acid. Treatment of LCAT with p-bromoacetylaminophenylarsenoxide blocks the subsequent incorporation of diisopropyl fluorophosphate and iodoacetamide and inactivates both cholesterol esterification and lecithin cleavage. These activities are not restored following 2,3-dimercapto-1-propanesulfonic acid treatment. However, the reduced cysteine thiols are regenerated and can catalyze cholesteryl arachidonate formation from arachidonyl-CoA. The control reagent, bromoacetylaniline, which lacks the sulfhydryl-reactive arsenical moiety, does not inactivate LCAT nor is this reagent incorporated into the LCAT protein. We conclude that the two catalytic cysteine residues of LCAT (Cys31 and Cys184) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by the bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

Arachidonic Acid↗

PubMed1987

Dihydrolipoamide dehydrogenase from Trypanosoma brucei. Characterization and cellular location.

Dihydrolipoamide dehydrogenase has been discovered in the bloodstream form of the eukaryotic African parasite, Trypanosoma brucei. The enzyme catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+ and exhibited a hyperbolic dependence of catalytic activity on the concentrations of both dihydrolipoamide and NAD+. Chemical modification with the tervalent arsenical reagent p-aminophenyldichloroarsine indicates the involvement in catalysis of a reversibly reducible disulphide bond. Plasma-membrane sheets were purified from T. brucei, and it was shown that virtually all the dihydrolipoamide dehydrogenase remained closely associated with this membrane preparation. T. brucei apparently lacks the 2-oxoacid dehydrogenase multienzyme complexes of which dihydrolipoamide dehydrogenase is usually an integral component. In the context of this absence, the possible function of trypanosomal dihydrolipoamide dehydrogenase is discussed, with particular reference to its cellular location in the plasma membrane.

Animals↗

PubMed1987

Spectroscopic characterization of thioredoxin covalently modified with monofunctional organoarsenical reagents.

Thioredoxin upon reduction with mercaptoethylamine was subjected to covalent modification by the monofunctional organoarsenical reagents H2NPhAsO and HO(CH2)4AsCl2. The degree of modification was monitored by the percentage loss in free thiol content as measured by the reaction with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid). The modification results in the formation of a stable 15-membered cyclic dithioarsenite ring that readily extrudes the arsenic moiety upon the addition of 2,3-dimercaptopropanol. The conformational effects of this modification were monitored by steady-state fluorescence and circular dichroism. On the basis of circular dichroic spectra, it appeared that the protein experiences no significant backbone conformational change from this modification. The degree of conformational change was found to be within the range observed upon reduction of the oxidized thioredoxin. Steady-state fluorescence revealed that the arsenicals caused strong quenching of the tryptophan fluorescence. Stern-Volmer titrations revealed that the quenching was a function of both the nature of the organic group and its covalent attachment to the "spatially close" thiols. The analysis of the spectroscopic results obtained with the arsenical reagents provides further insight into the nature of the conformational change that has been observed upon reduction of thioredoxin.

Arsenicals↗

PubMed1986

The reliability of activated partial thromboplastin time methods and the relationship to lipid composition and ultrastructure.

Wide variations in procoagulant properties, lipid composition and ultrastructure of five commonly used APTT methods have been demonstrated. Performance of the methods with a range of coagulation abnormalities has been ranked. Most of the reagents obtained a high score with one or more defects, but a low score with others. A consistent good ranking throughout was only observed with one reagent. The number of significant correlations between the reagents' procoagulant activities and lipid content confirms the view that the performance of an APTT method is largely dependent upon its lipid composition. Marked differences in concentration and distribution of phospholipids, fatty acids and neutral lipids were evident. The importance of the concentration of phosphatidyl serine in regulating the procoagulant activity of an APTT method has been demonstrated. Electron microscopy provides evidence of the contrasting composition of the reagents from the more discrete uniform liposomes present in the more reliable reagents, to more ill-defined components present in those reagents which performed less well. The study highlights the need for standardisation of the APTT.

Blood Coagulation Tests↗

PubMed1986

Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptible to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the--AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH--group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.

Acetylation↗

PubMed1986

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide.

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.

Amino Acid Sequence↗

PubMed1985

Measurements of carbon monoxide and nitrogen dioxide in British homes using unflued heating or cooking appliances.

Measurements of Carbon Monoxide (CO) and Nitrogen Dioxide (NO2) were made in three kitchens, two with gas cookers and one all electric, and two living rooms, one heated by a portable butane heater and the other by a paraffin stove. The measurements were taken whilst the homes were in normal use and the occupants kept a diary of when specific appliances were used. Measurements of NO2 were made simultaneously by chemiluminescence and by diffusion tubes. The accuracy of the diffusion tube measurements relative to the chemiluminescent standard was estimated as +/- 10% and the precision less than or equal to 300 ppb. hr. Results from diffusion tubes were used to indicate the distribution of NO2 throughout other rooms in the house. At the all electric house the CO and NO2 concentrations were very similar to the expected outdoor levels. In all the other houses NO2 levels exceeded typical outdoor concentrations measured in Central London and in the paraffin heated house and one gas cooking house median CO concentrations exceeded these measured at the curbside of a very busy road in Central London. The living room concentrations of NO2 where the paraffin heater was in use were elevated for long periods. Analysis of the decay of pollutant concentration in one kitchen yielded an effective deposition velocity for NO2 of 3 X 10(-5) m s-1. The magnitude of the CO and NO2 concentrations measured in this survey confirms that indoor air pollution should be taken into account when assessing overall public exposure to air pollution.

Carbon Monoxide↗

PubMed1984

The diagnosis of lupus anticoagulants by the activated partial thromboplastin time--the central role of phosphatidyl serine.

Liposomes of pure phospholipids were used in a modified APTT test system and the role of phosphatidyl serine (PS) in determining the sensitivity of the test system to the presence of lupus anticoagulants was assessed. Six consecutive patients with lupus anticoagulants and seven haemophiliacs with anticoagulants directed at specific coagulation factors, were studied. Increasing the concentration of phospholipid in the test system markedly reduced the sensitivity to lupus anticoagulants but had marginal effect on the specific factor inhibitors. The same effect was achieved when the content of PS alone was increased in a vehicle liposome of constant composition. The results suggest that the lupus anticoagulants can best be detected by a screening method using an APTT test with a reagent of low PS content. The use of a reagent rich in PS will largely abolish the lupus anticoagulant's effect on the APTT. An approach using the two different types of reagent may facilitate differentiation of lupus inhibitors from other types of anticoagulant.

Adult↗

PubMed1984

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase.

Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189]. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.

Alkylation↗

PubMed1983

Lipid class composition and heparin sensitivity in the activated partial thromboplastin time.

In an APTT reagent, prepared from purified lipids, the role of phosphatidyl serine (PS) in determining the sensitivity of the APTT test system to measurement of the effect of heparin in plasma has been evaluated. As the concentration of PS decreases sensitivity to heparin increases but procoagulant activity decreases. Dilution of the test liposome over a wide range (1 g/l to 30 mg/l) had a minimal effect on the clotting time. At levels below 30 mg/l, however, the amount of total lipid appeared to be rate limiting; a loss of procoagulant activity being paralleled by an increase in heparin sensitivity. Phosphatidyl inositol (PI) was not a satisfactory substitute for PS in the APTT method studied. The degree of unsaturation of test liposomes appeared to have no effect on either procoagulant activity or sensitivity to heparin at the lipid concentration employed. In the light of these findings, a more critical appraisal of the phospholipid components of APTT reagents should facilitate the development of more reliable reagents for heparin control. A further benefit of this type of approach should be a reduction in the acknowledged wide variations in sensitivity to heparin which exist between available APTT reagents.

Blood Coagulation Tests↗

PubMed1982

Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa.

An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns. The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions. The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium. The H. lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues. This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates. This enzyme represents a unique example of a thiol proteinase obtained from a fungal source.

Chromatography, Affinity↗

PubMed1982

The procoagulant activity of partial thromboplastin extracts: the role of phosphatidyl serine.

The role of phosphatidyl serine (PS) in the procoagulant activity of the APTT test has been studied further, using extracts of improved purity derived from a combination of high performance liquid chromatography and thin layer chromatography. The central role of PS in the APTT has been confirmed but the anticoagulant effect described in our previous report has been localised to contamination with phosphatidyl inositol (PI). A comparatively weak procoagulant activity was detected in purified PI- and lyso PS-containing liposomes and the former had an inhibitory action in mixtures diluted to less than 1 microgram/litre. This was enhanced by the presence of PS in combination experiments. Some correlation was noted between the degree of unsaturation of test liposomes and their procoagulant activity. The observations suggest that PS has a precise and unique role in the procoagulant activity expressed in this test system. PS possesses a strong negative charge and has two fatty acid groups. Other negatively charged phospholipids in this test system were less active. It appears, therefore, that the packing of PS into the lipid bilayer, which depends upon the length and degree of unsaturation of its fatty acid groups, is of considerable importance in establishing the surface topography essential for optimal procoagulant activity. Knowledge of the role played by various phospholipids in the procoagulant activity of the APTT reagent should enable improved standardisation and quality control in the future.

Blood Coagulation↗

PubMed1982

Purification and characterization of dihydrofolate reductase from Walker 256 carcinosarcoma.

Dihydrofolate reductase of Walker 256 carcinosarcoma has been purified to homogeneity by affinity chromatography. The enzyme reduced 28 mumol dihydrofolate (FAH2) . min-1 . mg protein-1 at 22 degrees C and pH 7.3. Km values with respect to FAH2 and NADPH were 21 and 29 mM, respectively. The IC50 (amount of inhibitor required for 50% loss of enzyme activity) values were 0.2 nM for MTX and aminopterin and 50 and 67 nM, respectively, for N10-formyl FA and triazinate (NSC-139105). The pH maximum is around pH 7.0 and the isoelectric point is 6.8. This reductase has an apparent molecular weight of 21 500. The N-terminal amino acid is valine and the comparison of the N-terminal 20 residues of this reductase shows very high sequence homology with other mammalian reductases. The enzyme contains two cysteine residues and one of these residues is not involved in catalysis. This reductase has four tryptophan residues and modification of one of these residues leads to loss of activity. The intrinsic circular dichroic (CD) spectrum of this reductase is very different from the CD spectra of reductase of Escherichia coli B and L1210/MTX. However, the CD spectra of the enzyme--substrate and enzyme--inhibitor complexes are very similar to that of the L1210/MTX enzyme. This suggests that the ligands may be constrained in similar conformation on the two enzymes. The fluorescence emission maximum at 314 nM when activated at 286 nM is considerably lower than other mammalian enzymes.

Animals↗

PubMed1981

Modification of the thiol residues of pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Activity modulation by the divalent thiol reagent p-aminophenylarsenoxide.

1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions. 2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity. 3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethyl-maleimide. Modification of one residue leads to loss of 40-60% of the enzyme activity in both the forward (NAD+ + NADPH leads to NADH + NADP+) and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction [Voordouw et al. (1980) Eur. J. Biochem. 107, 337-344] is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate. 4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x 10(5) M-1 at 25 degrees C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity of p-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex. 5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.

Arsenicals↗

PubMed1981

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.

The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.

Arsenicals↗

PubMed1980

Reactions of oxidizing and reducing radical probes with lipoamide dehydrogenase.

One-electron redox radicals generated by radiation--chemical methods have been reacted with the oxidized (E) form of pig heart lipoamide dehydrogenase. The reducing radicals eaq- and CO2-. and O2-. do not measurably inactivate the enzyme, whereas the oxidizing species .OH and Br2-. do. The CO2-. anion forms the semiquinone radical .EH on the millisecond time scale, whereas at longer times only EH2 is observed. Evidence suggests that Br2-. oxidizes adjacent sulfhydryl groups to form a disulfide in a manner similar to the reaction of Cu2+ ions. With .OH, destruction of the flavin adenine dinucleotide (FAD) moiety is responsible for at least 50% of the enzyme inactivation. This destruction appears to be a result of secondary reactions which transfer damage from remote initial sites of attack to the flavin. Pathways for migration of eaq- damage also appear to exist.

Bromides↗