Investigation of the Kinetics of Chemical Reactions

Perkampus, H.‐H.

doi:10.1007/978-3-642-77477-5_7

Cited by 1 publication

(2 citation statements)

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“…This evaluation is based upon the correlation of the time-dependent changes of the fluorescence measured at one emission wavelength (x-axis) with the fluorescence measured at another emission wavelength (yaxis). If the result of this correlation is a straight line, the reaction can be regarded as a homogenous process [14]. This procedure should be repeated with the data obtained at other wavelengths to avoid coincidental results.…”

Section: Fluorescence Changes Accompanying Sulfitolysismentioning

confidence: 99%

“…This treatment increased the fluorescence emission at 34.5 nm to the same value measured with the same amount of dithiothreitol-reduced wild-type thioredoxin (compare Fig. 1 Extinction diagrams of the type used in photochemistry have been applied to verify that the sulfitolysis of thioredoxin is a homogenous reaction [14]. This evaluation is based upon the correlation of the time-dependent changes of the fluorescence measured at one emission wavelength (x-axis) with the fluorescence measured at another emission wavelength (yaxis).…”

Section: Fluorescence Changes Accompanying Sulfitolysismentioning

confidence: 99%

See 1 more Smart Citation

Structure requirements for disulfide bridge sulfitolysis of oxidized Escherichia coli thioredoxin studied by fluorescence spectroscopy

Häberlein¹

1994

European Journal of Biochemistry

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Sulfitolysis of wild-type and four mutated Escherichia coli thioredoxins ([D26A]thioredoxin,[P34H] thioredoxin, [K36E]thioredoxin and endo-Arg3'"-thioredoxin) has been investigated at millimolar concentrations of sulfite in the absence of protein-denaturing agents by fluorescence spectroscopy. Sulfitolysis of the single disulfide bridge of these proteins is associated with an increase in fluorescence emissions at 345 nm. Evaluation of the fluorescence emission spectra revealed that sulfitolysis of thioredoxins is a homogenous process. The reactivities of the thioredoxins are determined by negatively charged (Asp26) or positively charged (Lys36) amino acid residues near the active site disulfide bridge.Thioredoxins are small and stable proteins (Mr 10000-12000) that have been isolated and characterized from many different microbial organisms, plants and animals [ 11. The active site of thioredoxin (-Trp-Cys-Gly-Pro-Cys-) is highly conserved and contains two redox-active cysteine residues separated by a glycine and a proline residue [2]. Oxidized thioredoxin forms a disulfide bridge that is readily reduced to the dithiol because of its exposed position on an edge of the globular protein [2]. The reduced thioredoxins are powerful oxido-reductases involved in many essential biochemical processes such as deoxyribonucleotide biosynthesis, hormone activation or inactivation, and the lighudark regulation of chloroplast enzymes [31.We have reasoned that the sulfitolysis of oxidized thioredoxin is a key chemical event explaining the phytotoxic action of sulfur dioxide upon plants [4, 51. In this process, the disulfide bridge of oxidized thioredoxin is cleaved by sulfite ions at physiological, millimolar concentrations, forming an inactive S-sulfonyl thioredoxin (Scheme 1). In previous experiments we measured sulfitolysis by labeling the cysteine residue generated in close proximity to the S-sulfonyl group with iod0[2-~H]acetic acid [6]. This method is limited by the accessibility of this cysteine residue for the alkylating agent. In the case of Escherichia coli thioredoxin it has been shown that carboxymethylation of both cysteine residues (Cys32 and Cys35) with iodoacetic acid is incomplete. The alkylation of Cys35 is, in particular, sterically hindered [7]. If we assume that sulfitolysis occurs at Cys32 and that the less accessible Cys35 is the one residue must be alkylated, the observed yield of E. coli thioredoxin sulfitolysis in the presence of 5 mM sulfite ions, measured by the iodoacetic acid Scheme 1. Formation of S-sulfonyl thioredoxin.Thioredoxin S-sulfonyl thioredoxin alkylation method (less than 5 % ) , may represent only a proportion of the actual yield [6]. To avoid such a limitation, we took advantage of the fact that the fluorescence of E. coli thioredoxin increases significantly upon reductive opening of the disulfide bridge [S], and that sulfitolytic cleavage induces a similar fluorescence change [6]. In this study, the sulfitolysis reaction of native and mutant E. coli thioredoxins measured by fluoresc...

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Section: Fluorescence Changes Accompanying Sulfitolysismentioning

confidence: 99%

Section: Fluorescence Changes Accompanying Sulfitolysismentioning

confidence: 99%