[8] Reassessment of Ellman's reagent

Riddles, Peter W.; Blakeley, Robert L.; Zerner, Burt

doi:10.1016/s0076-6879(83)91010-8

Cited by 1,099 publications

(851 citation statements)

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“…Purified BMH-, DBP-, and K70C mustard-crosslinked dimers, along with purified monomers from BMH, MCA, and mustard reactions, were subjected to a standard Ellman's assay (Riddles et al, 1983) to quantify the extent of modification at cysteine residues. Wild-type and untreated cysteine standards were also assayed as controls.…”

Section: Resultsmentioning

confidence: 99%

“…Ellman's assay DBP-and BMH-purified crosslinked dimers and K70C mustard crosslinked dimer were subjected to a standard Ellman's assay (Riddles et al, 1983) to quantify the extent of crosslinking at cysteine residues. Also, BMH-and mustard-purified monomers were subjected to an Ellman's assay to quantify the extent of cysteine modification.…”

Section: Mca Modificationmentioning

confidence: 99%

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Chemically crosslinked protein dimers: Stability and denaturation effects

Byrne

Stites

1995

Protein Science

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Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; I ,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethy1)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCI) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, e-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Mca Modificationmentioning

confidence: 99%

Chemically crosslinked protein dimers: Stability and denaturation effects

Byrne

Stites

1995

Protein Science

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“…Tryptophan was determined spectrophotometrically (7). Free sulfhydryl groups were determined with Ellman's reagent (25).…”

Section: Amino Acid Analysismentioning

confidence: 99%

Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli

Biedermann

Jepsen

Riise

et al. 1989

Carlsberg Research Communicati

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The primary structure and physical chemical properties were determined of a nuclease expressed and secreted by Escherichia colt. The plasmid p403-SD2 carried a DNA sequence isolated from Serratia marcescens encoding the enzyme. During cultivation of the E. colt cells, 85% of the enzyme was released to the growth medium. The enzyme was purified and exhibited a single band with a molecular weight about 30,600 daltons on SDS-PAGE similar to nuclease isolated from S. marcescens. The amino acid composition and the amino acid sequence determined directly confirmed the primary structure of 245 amino acids predicted from the DNA sequence, and, in addition, the two disulfide bridges were assigned. Several physical chemical properties were examined. The ability of the enzyme to cross the outer membrane is proposed to depend upon the formation of the proper structures during the folding process.

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“…Vacuum desiccation over calcium sulfate was insufficient to remove this water. PEG-a-Cys was not heated to drive off the water, as there was concem that at elevated temperatures the presence of water and possibly trace arnounts of DMAP from the synthesis rnight result in base-catalyzed hydrolysis of the ester or decomposition of the Ellman's reagent portion of the molecule (Riddles et al, 1983). As it was intended that PEG-a-Cys would be dissolved in aqueous solution before reaction with its target peptide, the presence of a small amount of bound water was not deemed to be of great concem.…”

Section: Svnthesismentioning

confidence: 99%