The interaction of copper(II/I) complexes of a few 2,9-dimethyl-1,10-phenanthrolines with calf thymus DNA has been investigated using absorption and circular dichroic spectral and electrochemical techniques and viscometry. The observation of the usual hypochromism and the novel hyperchromism in the absorption spectra of [CuI(bcp)2]+ [bcp = 2,9-dimethyl-4,7-diphenyl-1,10- phenanthroline] and [CuI(dpsmp)2]3- [dpsmp2- = 2,9-dimethyl-4,7-bis(sulfonatophenyl)-1,10-phenanthroline] respectively in the presence of DNA and the increase in viscosity of DNA at low loadings of both these complexes have been interpreted in terms of bridging of a pair of DNA duplexes by the complex species. These tetrahedral copper(I) complexes, which lack minor groove binding because of substituents at the 4- and 7-positions of phen ring, are efficient in bridging the duplexes. The electrochemical behaviors of [CuI(dmp)2]+ [dmp = 2,9-dimethyl-1,10-phenanthroline] and [CuI(bcp)2]+ bound to DNA have been compared with that of the analogous sulfonated complex [Cu(dpsmp)2]2-/3-. The DNA binding constants determined reveal that dpsmp2- complex is engaged in DNA binding less intimately than the bcp complex. While Coulombic interactions are clearly more important than other types of interactions for the former, nonclassical hydrophobic interactions for the latter. The Hill analysis of the absorbance data obtained as a function of added DNA reveals Hill coefficients greater than unity, which may be construed as evidence for cooperative binding of the copper complexes to B-DNA.

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification, and quantification of growing importance. Amongst free cysteines, the bulk of modifications occur on a subset of cysteines that are more reactive. These exist as thiolate anions at physiological pH because of their surrounding electrostatic environment. Reagents with iodoacetamide active groups can be used to selectively label these reactive thiols with a high degree of selectivity. Thiol adducts can be detected by the failure to label with iodoacetamide or other reagents; restoration of labeling by specific reducing agents (eg. ascorbate or glutaredoxin), can be used to detect reversible S-nitroso and S-glutathione adducts. These adducts also may be detected with radiolabels and antibodies. S-glutathiolation in response to physiological stimuli may be detected in cells and tissues with glutathione ester labeled with biotin. Mass spectrometry can identify thiol modifications with precision, and with isotope-coded affinity tags, used to quantify modification of specific thiols. Combinations of these methods increase sensitivity and specificity, and enable quantification and precise identification of thiol modifications that occur under physiological and pathological conditions.

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.

An approach is described for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant-sensitive, was chosen as an experimental model. ICAT-labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected (or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant-sensitive thiol residue of cysteine-283 in creatine kinase, providing proof of principle that an ICAT-based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols. Regulation of cellular homeostasis through post-translational modification of proteins is one of the major responses to oxidative and nitrosative stress (1). Proteins containing cysteine thiol groups are particularly susceptible to oxidation by free radicals, electrophilic molecules, and nitric oxide donors (2, 3). One or more reduced thiol groups are essential for the function of many proteins. Oxidation of these critical thiol groups can increase or decrease the activity of these proteins and represents not only a major mechanism of normal cell signaling via S-nitrosation (4) or S-glutathiolation (5), but also a mechanism by which disease and aging interferes with protein function by irreversible thiol oxidation (6). Thus, it is essential to identify the proteins containing Cys residues and their relative sensitivity to oxidation. As a step in the development of a proteomic approach to identify post-translational modification of Cys residues in proteins involved in redox signaling or those affected by disease, this report describes a method to identify and quantify oxidant-sensitive protein thiols by mass spectrometric peptide fingerprinting.The procedure is based on the fact that oxidized Cys residues are not susceptible to modification by iodoacetamide (IAM) 1 analogues (7). Isotope-coded affinity tag (ICAT) reagents that are IAM analogues have been used extensively in quantitative proteomics to evaluate the abundance of expressed proteins (8). The ICAT approach is based on affinity tag targeting of free cysteines in proteins that are labeled after the proteins are isolated under strong reducing conditions. This study was designed to determine the feasibility of using the acid-cleavable IAM-based ICAT reagent (catalogue no. 4337335; Applied Biosystems, Foster City, CA) to quantitate the extent of thiol oxidation under nonreducing conditions. The principle of the ICAT app...

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