Introduction of sulfhydryl groups into proteins at carboxyl sites.
AuthorsLin CM, Mihal KA, Krueger RJ
JournalBiochim Biophys Acta
PubMed ID2160279
'A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. ... More
Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.
AuthorsSmith DJ, Maggio, ET, Kenyon GL
JournalBiochemistry
PubMed ID163643
'New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with ... More
An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.
AuthorsHansen RE, Winther JR,
JournalAnal Biochem
PubMed ID19664585
'This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we ... More
Measurement of cyst(e)amine in physiological samples by high performance liquid chromatography.
AuthorsGarcia RA, Hirschberger LL, Stipanuk MH
JournalAnal Biochem
PubMed ID3394941
'Two methods for measurement of cyst(e)amine in physiological samples are described. One method involves reduction of disulfides present in the sample with tributylphosphine, reversed phase chromatography of thiols, and electrochemical detection of cysteamine and other thiols. The other method involves reduction of disulfides with dithiothreitol, derivatization of thiols with 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin, ... More
Chemical reduction of disulfides.
AuthorsJocelyn PC
JournalMethods Enzymol
PubMed ID3657541
Photodynamic therapy and cell death pathways.
AuthorsKessel D, Oleinick NL,
JournalMethods Mol Biol
PubMed ID20552338
Photodynamic therapy (PDT) is the term used to describe the irradiation of photosensitized cells or tissue with phototoxic consequences. This process can result in the rapid initiation of not only apoptosis, an irreversible death pathway, but also autophagy. The procedures described here are designed to characterize the correlation between the ... More
Biotin-protein bond: instability and structural modification to provide stability for in vivo applications.
AuthorsMock DM, Bogusiewicz A,
JournalMethods Mol Biol
PubMed ID18287661
Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin-protein bonds that are susceptible ... More
In-gel detection of S-nitrosated proteins using fluorescence methods.
AuthorsKettenhofen NJ, Wang X, Gladwin MT, Hogg N,
JournalMethods Enzymol
PubMed ID18554529
Gel-based detection of protein S-nitrosothiols has relied on the biotin-switch method. This method attempts to replace the nitroso group with a biotin label to allow detection and isolation of S-nitrosated proteins and has been used extensively in the literature. This chapter describes a modification of this method that differs from ... More
Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.
AuthorsOdom OW, Picking WD, Hardesty B
JournalBiochemistry
PubMed ID1703007
The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more ... More
Selective enrichment of thiophosphorylated polypeptides as a tool for the analysis of protein phosphorylation.
AuthorsKwon SW, Kim SC, Jaunbergs J, Falck JR, Zhao Y,
JournalMol Cell Proteomics
PubMed ID12734387
A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5'-O-(thiotriphosphate) (ATPgammaS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity ... More
Efficient site-specific labeling of proteins via cysteines.
AuthorsKim Y, Ho SO, Gassman NR, Korlann Y, Landorf EV, Collart FR, Weiss S,
JournalBioconjug Chem
PubMed ID18275130
Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using ... More
Sample preparation for peptide mapping--A pharmaceutical quality-control perspective.
AuthorsLundell N, Schreitmüller T
JournalAnal Biochem
PubMed ID9887211
In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the experimental procedure are therefore different than in the case of determination of an unknown protein structure. It is here recognized that a peptide-mapping method for ... More
Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.
AuthorsRhee SS, Burke DH
JournalAnal Biochem
PubMed ID14715294
We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] ... More
Dithiothreitol treatment of Madin-Darby canine kidney cells reversibly blocks export from the endoplasmic reticulum but does not affect vectorial targeting of secretory proteins.
AuthorsLösch A, Koch-Brandt C
JournalJ Biol Chem
PubMed ID7744793
Addition of dithiothreitol (DTT) to the culture medium of Madin-Darby canine kidney (MDCK) cells blocks transport of newly synthesized gp80 (clusterin, apolipoprotein J), a soluble marker protein for apical exocytosis in this epithelial cell line. In cells treated with DTT during pulse labeling, gp80 is retained in the endoplasmic reticulum. ... More
Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase.
AuthorsAnderson PM, Korte JJ, Holcomb TA, Cho YG, Son CM, Sung YC
JournalJ Biol Chem
PubMed ID8195141
Reaction of the single cysteine residue in each subunit of cyanase with certain SH reagents gives an active decameric derivative that dissociates reversibly to an inactive dimer derivative (Anderson, P. M., Johnson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1988) J. Biol. Chem. 263, ... More
A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry.
AuthorsGetz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR
JournalAnal Biochem
PubMed ID10452801
The newly introduced sulfhydryl reductant tris(2-carboxyethyl)phosphine (TCEP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemistry, using the motor enzyme myosin as an example protein. The reductants equally preserve myosin's enzymatic activity, which is sensitive to sulfhydryl oxidation. ... More
Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity.
AuthorsAsahi M, Fujii J, Suzuki K, Seo HG, Kuzuya T, Hori M, Tada M, Fujii S, Taniguchi N
JournalJ Biol Chem
PubMed ID7673130
S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC50 of SNAP for GPx was 2 microM at 1 h of incubation and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue ... More