Regionally Impaired Redox Homeostasis in the Brain of Rats Subjected to Global Perinatal Asphyxia: Sustained Effect up to 14 Postnatal Days.

The present report evaluates the effect of global perinatal asphyxia on several parameters of oxidative stress and cell viability in rat brain tissue sampled at an extended neonatal period up to 14 days, a period characterised by intensive neuritogenesis, synaptogenesis, synaptic consolidation, pruning and delayed cell death. Perinatal asphyxia was induced by immersing foetus-containing uterine horns removed by a caesarean section from on term rat dams into a water bath at 37 °C for 21 min. Asphyxia-exposed and sibling caesarean-delivered foetuses were manually resucitated and nurtured by surrogate dams for 1 to 14 postnatal (P) days. Brain samples (mesencephalon, telencephalon and hippocampus) were assayed for glutathione (reduced and oxidated levels; spectrophotometry), tissue reducing capacity (potassium ferricyanide reducing assay, FRAP), catalase (the key enzyme protecting against oxidative stress and reactive oxygen species, Western blots and ELISA) and cleaved caspase-3 (the key executioner of apoptosis, Western blots) levels. It was found that global PA produced a regionally specific and sustained increase in GSSG/GSH ratio, a regionally specific decrease in tissue reducing capacity and a regionally and time specific decrease of catalase activity and increase of cleaved caspase-3 levels. The present study provides evidence for regionally impaired redox homeostasis in the brain of rats subjected to global PA, an effect observed up to P14, mainly affecting mesencephalon and hippocampus, suggesting a sustained oxidative stress after the posthypoxia period. The oxidative stress observed postnatally can in part be associated to a respiratory apneic-like deficit, since there was a statistically significant decrease in respiration frequency in AS compared to CS neonates, also up to P14, together with the signs of a decreased peripheral blood perfusion (pink-blue skin colour in AS, compared to the pink colour observed in all CS neonates). It is proposed that PA implies a long-term metabolic insult, triggered by the length of hypoxia, the resuscitation/reoxigenation manoevres, but also by the developmental stage of the affected brain regions, and the integrity of cardiovascular and respiratory physiological functions, which are fundamental for warrantying a proper development.

Antioxidant properties in a non-polar environment of difluoromethyl bioisosteres of methyl hydroxycinnamates.

OBJECTIVES: Many natural antioxidants have poor pharmacokinetic properties that impair their therapeutic use. For hydroxycinnamic acids (HCAs) and other phenolic antioxidants, their major drawback is their low lipophilicity and a rapid metabolism. The difluoromethyl group may be considered as a 'lipophilic hydroxyl' due to its hydrogen bond donor and acceptor properties; this prompted us to assess it as a bioisosteric replacement of a phenolic hydroxyl for increasing the lipophilicity of HCAs. METHODS: Six difluoromethyl-substituted methyl cinnamates (4a-c, 5a-c) related to caffeic acid were synthesized and their antioxidant activity evaluated by chemical (FRAP, DPPH scavenging, inhibition of ß-carotene bleaching, at 1-200 µm), electrochemical (differential pulse voltammetry, cyclic voltammetry) and cell-based (inhibition of lipid peroxidation in erythrocytes, at 1 and 50 µm) assays. KEY FNDINGS: Analogues 4a-c and 5a-c were inactive in FRAP and DPPH assays and only those containing a free phenolic hydroxyl (4a and 5a) exhibited electrochemical activity although with high redox potentials. Compounds 4a,b and 5a,b were active in the inhibition of ß-carotene bleaching assay and all analogues inhibited lipid peroxidation in the human erythrocytes assay. CONCLUSIONS: Lipophilic difluoromethyl-substituted cinnamic esters retain radical scavenging capabilities that prove useful to confer antioxidant properties in a non-polar environment.

Adherence to standard precautions from the standpoint of the Health Belief Model: the practice of recapping needles / Adesão às precauções padrão sob o prisma do Modelo de Crenças em Saúde: a prática de reencapar agulhas

The aim of this study was to applythe Health Belief Model to explain the adherence to the recommendation not to recap needles by dentists and dental assistants of the public health system in a municipality in the State of São Paulo. A questionnaire validated and adapted for the oral health area was used, which included variables related to the frequency of recapping and health beliefs using Likert-type scales. The relationship between beliefs and adherence to the recommendation not to recap needles was obtained by regression analysis. Of all the professionals in this study (n=79), the majority (83.5%) reported recapping needles at least once in the last month. Through regression analysis, it was observed that the relationship between the beliefs described by the model and the attitude whether or not to follow the recommendation not to recap needles was explained by a lower perception of psychological barriers and a greater perception of stimuli not to recap needles. The conclusion reached is that the acceptance of recommendations to prevent working accidents with biological material was explained by some dimensions of the Health Belief Model, enabling discussion about reformulation of training offered to professionals of the public health system.

Objetivou-se neste estudo aplicar o Modelo de Crenças em Saúde a fim de explicar a adesão à recomendação de não reencapar agulhas por cirurgiões-dentistas e auxiliares de saúde bucal da rede pública de um município paulista. Utilizou-se um questionário validado e adaptado para a área de saúde bucal, que contemplava variáveis relativas à frequência do reencape e crenças em saúde, por meio de escalas tipo Likert. A relação entre as crenças e a adesão à recomendação de não reencapar agulhas foi obtida por meio da análise de regressão. Da amostra de profissionais obtida por adesão ao estudo (n = 79), a maioria (83,5%) relatou ter reencapado agulhas pelo menos alguma vez no último mês. Por meio da análise de regressão, foi observado que a relação entre as crenças descritas pelo modelo e a atitude de aderir ou não à recomendação de não reencapar agulhas foi explicada por uma menor percepção de barreiras psicológicas e por uma maior percepção de estímulos para não reencapar agulhas. Conclui-se que a aceitação das recomendações para prevenir acidentes do trabalho com material biológico foi explicado por algumas dimensões do Modelo de Crenças em Saúde, possibilitando a discussão sobre a reformulação de capacitações oferecidas para profissionais do sistema público de saúde.

A thiophene-modified screen printed electrode for detection of dengue virus NS1 protein.

A thiophene-modified screen printed electrode (SPE) for detection of the Dengue virus non-structural protein 1 (NS1), an important marker for acute phase diagnosis, is described. A sulfur-containing heterocyclic compound, the thiophene was incorporated to a carbon ink to prepare reproducible screen printed electrodes. After cured, the thiophene SPE was coated by gold nanoparticles conjugated to Protein A to form a nanostrutured surface. The Anti-NS1 antibodies immobilized via their Fc portions via Protein A, leaving their antigen specific sites free circumventing the problem of a random antibodies immobilization. Amperometric responses to the NS1 protein of dengue virus were obtained by cyclic voltammetries performed in presence of ferrocyanide/ferricyanide as redox probe. The calibration curve of immunosensor showed a linear response from 0.04 µg mL(-1) to 0.6 µg mL(-1) of NS1 with a good linear correlation (r=0.991, p<0.05). The detection limit (0.015 µg mL(-1) NS1) was lower than conventional analytical methods. In this work, thiophene monomers incorporated in the carbon ink enhanced the electroanalytical properties of the SPEs, increasing their reproducibility and sensitivity. This point-of-care testing represents a great potential for use in epidemic situations, facilitating the early diagnosis in acute phase of dengue virus.

A carbon nanotube screen-printed electrode for label-free detection of the human cardiac troponin T.

Label-free immunosensor based on amine-functionalized carbon nanotubes screen-printed electrode is described for detection of the cardiac troponin T, an important marker of acute myocardial infarction. The disposable sensor was fabricated by tightly squeezing an adhesive carbon ink containing carbon nanotubes onto a polyethylene terephthalate substrate forming a thin film. The use of carbon nanotubes increased the reproducibility and stability of the sensor, and the amine groups permitted nonrandom immobilization of antibodies against cardiac troponin T. Amperometric responses were obtained by differential pulse voltammetry in presence of a ferrocyanide/ferricyanide redox probe after troponin T incubation. The calibration curve indicated a linear response of troponin T between 0.0025 ng mL(-1) and 0.5 ng mL(-1), with a good correlation coefficient (r=0.995; p<0.0001, n=7). The limit of detection (0.0035 ng mL(-1) cardiac troponin T) was lower than any previously described by immunosensors and was comparable with conventional analytical methods. The high reproducibility and clinical range obtained using this immunosensor support its utility as a potential tool for point-of-care acute myocardial infarction diagnostic testing.

Reactivity of iron(II)-bound nitrosyl hydride (HNO, nitroxyl) in aqueous solution.

The reactivity of coordinated nitroxyl (HNO) has been explored with the [Fe(II)(CN)(5)HNO](3-) complex in aqueous medium, pH 6. We discuss essential biorelevant issues as the thermal and photochemical decompositions, the reactivity toward HNO dissociation, the electrochemical behavior, and the reactions with oxidizing and reducing agents. The spontaneous decomposition in the absence of light yielded a two-electron oxidized species, the nitroprusside anion, [Fe(II)(CN)(5)NO](2-), and a negligible quantity of N(2)O, with k(obs)≈5×10(-7)s(-1), at 25.0°C. The value of k(obs) represents an upper limit for HNO release, comparable to values reported for other structurally related L ligands in the [Fe(II)(CN)(5)L](n-) series. These results reveal that the FeN bond is strong, suggesting a significant σ-π interaction, as already postulated for other HNO-complexes. The [Fe(II)(CN)(5)HNO](3-) ion showed a quasi-reversible oxidation wave at 0.32 V (vs normal hydrogen electrode), corresponding to the [Fe(II)(CN)(5)HNO](3-)/[Fe(II)(CN)(5)NO](3-),H(+) redox couple. Hexacyanoferrate(III), methylviologen and the nitroprusside ion have been selected as potential oxidants. Only the first reactant achieved a complete oxidation process, initiated by a proton-coupled electron transfer reaction at the HNO ligand, with nitroprusside as a final oxidation product. Dithionite acted as a reductant of [Fe(II)(CN)(5)HNO](3-), in a 4-electron process, giving NH(3). The high stability of bound HNO may resemble the properties in related Fe(II) centers of redox active enzymes. The very minor release of N(2)O shows that the redox conversions may evolve without disruption of the FeN bonds, under competitive conditions with the dissociation of HNO.

Evaluation of Magainin I interactions with lipid membranes: an optical and electrochemical study.

Most antimicrobial peptides (AMPs) have shown clear activity related to the disruption of lipid bilayers. In order to improve knowledge of this subject, the interaction of Magainin I (MagI) with phospholipid layers (PLs), uncoated or coated with synperonic (Synp), was studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques. MagI peptide was immobilized on gold electrode via a self-assembling monolayer obtained from liposomes and liposomes covered by Synp. MagI induces pores in the supported lipid membranes, which are reflected in an increased amperometric-response and also a decreased electron-transfer resistance (R(CT)). In addition, MagI showed a significant interaction with the PL-Synp-modified gold electrode, but MagI showed a reliable contact with the PL-modified gold electrode, leading to a decrease in the relative resistance charge transfer value of -17.38%. Our results demonstrated that Synp acts as a membrane sealant after exposure of the lipid membrane to MagI. A parallel reaction model was proposed for the interaction of MagI and a hybrid layer that result in a complex bimolecular interaction. In short, the importance of triblock copolymer to stabilize liposomes for future applications as drug delivery systems for MagI was demonstrated.

Coordination forces between lipid bilayers produced by ferricyanide and Ca2+.

Attractive forces usually invoked to take place in membrane-membrane contact in aggregation are hydrogen bonding cross-linkings and hydrophobic interactions between opposing surfaces. However, little is known in relation to the presence of coordination forces in the membrane-membrane interaction. These are understood as those that may be favoured by the formation or the participation of coordination complexes between surface specific groups. In this work, we have analyzed the formation of this type of aggregates between phosphatidylcholine vesicles mediated by a coadsorption of ferricyanide and Ca(2+) ions to the interface. The results obtained by surface potential measures, optical and electronic microscopy, FTIR and (1)H NMR spectroscopies indicate that ferricyanide [Fe(CN)(6)](3-) but not of ferrocyanide [Fe(CN)(6)](4-) can form the complex when Ca(2+) has been adsorbed previously to the membrane surface. In this condition, the anion is likely to act as a bridge between two opposing membranes causing a tight aggregation in which geometry and the polarizability of the ligands to Fe(3+) play a role.

Assessing the effect of oxygen and microbial inhibitors to optimize ferricyanide-mediated BOD assay.

Methods for short-term BOD analysis (BOD(st)) based on ferricyanide mediator reduction have succeeded in overcoming some problems associated with the standard BOD test analysis (BOD(5)) such as long-term incubations (5 days), the need to dilute samples and low reproducibility. Here we present a bioassay where a Klebsiella pneumoniae environmental strain successfully reduces ferricyanide without de-aeration of the samples with linear BOD(5) ranges between 30 and 500 mg L(-1) or 30 and 200 mg L(-1), using glucose-glutamic acid solution (GGA) or OECD standards respectively. We further propose a new assay termination solution that allows higher reproducibility and standardization of the cell-based assay, employing formaldehyde (22.7 g L(-1)) or other compounds in order to stop ferricyanide reduction without affecting the amperometric detection and therefore replace the centrifugation step normally used to stop microbial-driven reactions in ferricyanide-mediated bioassays. These improvements led to an accurate determination of real municipal wastewater samples.

Redox-active tyrosine residue in the microcin J25 molecule.

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H2O2 was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.

A critical evaluation of a flow-cell based on a liquid core waveguide for chemiluminescence measurements.

Liquid-core waveguides (LCWs), devices that constrain the emitted radiation minimizing losses during the transport, are an alternative to maximize the amount of detected radiation in luminescence. In this work, the performance of a LCW flow-cell was critically evaluated for chemiluminescence measurements, by using as model the oxidation of luminol by hydrogen peroxide or hypochlorite. An analytical procedure for hypochlorite determination was also developed, with linear response in the range 0.2-3.8 mg/L (2.7-51 micromol/L), a detection limit estimated as 8 microg/L (0.64 micromol/L) at the 99.7% confidence level and luminol consumption of 50 microg/determination. The coefficients of variation were 3.3% and 1.6% for 0.4 and 1.9 mg/L ClO(-), respectively, with a sampling rate of 164 determinations/h. The procedure was applied to the analysis of Dakin's solution samples, yielding results in agreement with those obtained by iodometric titration at the 95% confidence level.

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane.

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5mM ferrocyanide was almost completely inhibited after consumption of 3.8-18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.

Spectrophotometric determination of phenylephrine hydrochloride in pharmaceuticals by flow injection analysis exploiting the reaction with potassium ferricyanide and 4-aminoantipyrine.

This paper presents a method for the determination of phenylephrine hydrochloride in pharmaceuticals by spectrophotometric flow injection analysis exploiting the reaction with potassium ferricyanide and 4-aminoantipyrine, which leads to the formation of a condensation product with strong absorptivity at 500 nm. The linear dynamic range was between 0.95 and 9 mg/L, with a limit of detection of 0.2 mg/L and a sampling throughput of 120 samples per hour. The method was applied to eyewashes and nasal decongestant liquid medicines.

Histochemistry of the cuticle from proventriculus in stingless bee, Melipona quadrifasciata anthidioides (Hymenoptera, Apidae).

Histochemical study of the proventriculus from stingless bee Melipona quadrifasciata anthidioides revealed for the first time the presence of cysteine-rich proteins in the cuticle that covers the bulb of the proventriculus and its associated hair-like process. The presence of tyrosine-rich proteins was observed in the same structure, but not in hair-like projections. The cuticle of the crop and a small portion of the proventriculus contained no acid carbohydrates or fats and small amounts of neutral carbohydrates. These results are discussed in relation to different composition of insect cuticle depending on its different functions.

Modificación de un método para colinesterasa plasmática empleando la reducción del ferricianuro como indicador / Modification in the method for serum cholinesterase in ferricyanide reduction how indicatory

Se describe la modificación de un método para la cuantificación de colinesterasa plasmática mediante monitoreo continuo empleando ferricianuro como indicador. La tiocolina liberada de la propioniltiocolina reduce el ferricianuro a ferrocianuro y la disminición de absorbancia a 405 nm es proporcional a la actividad de la enzima. Las precisiones día a día para muestras con valores de colinesterasa bajos y altos mostraron coeficientes de variación de 3,2 y 1,2 por ciento y en un mismo día de 1,1 y 0,52 por ciento respectivamente. La bilirrubina no presenta una interferencia importante y cada 50mg/dL de hemoglobina producen una interferencia de -4 por ciento. El reactivo de ferricianuro almacenado en botella ámbar es estable por al menos 3 meses a temperatura ambiente y 6 meses a 4-8 §C. Al comparar los resultados con un método que emplea la reacción de Ellman se obtuvo una ecuación de regresión lineal de Y = 1,22 (X) - 732, con un coeficiente de correlación (r) de 0,988 y una desviación estándar sobre la línea de regresión (Sy/x) de 378 U/L. (Rev Cost Cienc Med 1999; 20(1,2): 17-27) PALABRAS CLAVE: Colinesterasa sérica, organofosfatos, carbamatos, pesticidas, métodos colorimétrico, toxicología

Chemical and photochemical generated carbon-centered radical intermediate and its reaction with desoxyribonucleic acid.

The possible action of carbon-centered radicals in promoting damage to DNA is explored by generation of the 2-phenylethyl radical. The radical is generated during oxidation of phenelzine (2-phenylethyl hydrazine) by ferricyanide, as well as optically from phenylpropionic acid. Covalent binding of the 2-phenylethyl radical to DNA is suggested by studies with the plasmid pBR 322 DNA. Other sensitive techniques used to study DNA damage were the interaction with formaldehyde at 60 degrees C and the fluorescence of DNA-Tb(III) and DNA-DAPI complexes. Theoretical MNDO calculations indicated a preferential attack at position 8 of the guanine residues. This study shows that the 2-phenylethyl radical is able to induce primary effects on nucleic acid structure, leading to alkylated products, especially at purine rings.

Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding.

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.

Intravenous infusions of nitroprusside, dobutamine, and nitroglycerin are compatible.

It is now customary practice to couple separately metered infusions via a manifold to a common catheter that enters the patient. Nitroprusside, however, is considered incompatible with all other medications. Critically ill patients who require multiple infusions of vasoactive and inotropic medications would benefit if physicians had additional information regarding compatibility of nitroprusside with other commonly used infusions. Utilizing high-performance liquid chromatography, the authors investigated the physical and chemical compatibility of nitroprusside, dobutamine, and nitroglycerin in solutions of 5% dextrose or 0.9% NaCl at clinically relevant concentrations. All drugs were present within the guidelines of the U.S. Pharmacopeia (+/- 10%) over 24 h in NaCl, but nitroglycerin degraded over 24 h when the three drugs were mixed in dextrose. We recommend diluting these medicines in NaCl when mixtures of them would exist for greater than 4 h.

Correlation between growth in antibiotic-medium and hemolytic activity of group C and G streptococci.

The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin O (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.

Cysteine labeling studies of beef heart aconitase containing a 4Fe, a cubane 3Fe, or a linear 3Fe cluster.

The reactivity of cysteines following cluster destruction by iron chelation was investigated for [4Fe-4S]2+ and cubane [3Fe-4S]+ beef heart aconitase. When the chelator orthobathophenanthroline disulfonate was used, the formation of sulfur-sulfur bonds and the retention of inorganic sulfur from the cluster was observed. For both the 4Fe and 3Fe forms of aconitase, the two cysteines in peptide 7, the cysteine in peptide 3, and the cysteine in peptide 2 were found as the primary constituents of sulfur-sulfur bonds (the peptide sequences and nomenclature are from Plank, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189). Three of these four cysteines (peptides 3 and 7) correlated with those proposed to be cluster ligands recently determined by x-ray crystallography (Robbins, A. H. and Stout, C. D. (1989) Proteins, in press; Robbins, A. H., and Stout, C. D.,, (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) for pig heart aconitase. A mechanism is proposed whereby the greater affinity of orthobathophenanthroline disulfonate for Fe2+ relative to Fe3+ shifts the equilibrium toward reduction of ferric iron through sulfur-sulfur bond formation at the cluster site. Aconitase which has been oxidized with ferricyanide and from which the cluster iron has been removed by EDTA has been shown to have two di- or polysulfides (Kennedy, M. C., and Beinert, H. (1988) J. Biol. Chem. 263, 8194-8198). The cysteines found in the sulfur-sulfur bonds generated by this treatment also were predominantly those from peptides 3 and 7. In addition, the putative thiol ligands for the linear [3Fe-4S]+ cluster of aconitase are reported. The four cysteines of peptides 7 and 9 (two in each peptide) were found to be protected by the cluster from alkylation when the protein was denatured. The difference in the ligands between the cubane and linear forms indicates that a specific thiol exchange occurs during the conversion.