Protein thiol oxidation does not change in skeletal muscles of aging female mice.

Oxidative stress caused by reactive oxygen species is proposed to cause age related muscle wasting (sarcopenia). Reversible oxidation of protein thiols by reactive oxygen species can affect protein function, so we evaluated whether muscle wasting in normal aging was associated with a pervasive increase in reversible oxidation of protein thiols or with an increase in irreversible oxidative damage to macromolecules. In gastrocnemius muscles of C57BL/6J female mice aged 3, 15, 24, 27, and 29 months there was no age related increase in protein thiol oxidation. In contrast, there was a significant correlation (R (2) = 0.698) between increasing protein carbonylation, a measure of irreversible oxidative damage to proteins, and loss of mass of gastrocnemius muscles in aging female mice. In addition, there was an age-related increase in lipofuscin content, an aggregate of oxidised proteins and lipids, in quadriceps limb muscles in aging female mice. However, there was no evidence of an age-related increase in malondialdehyde or F2-isoprostanes levels, which are measures of oxidative damage to lipids, in gastrocnemius muscles. In summary, this study does not support the hypothesis that a pervasive increase in protein thiol oxidation is a contributing factor to sarcopenia. Instead, the data are consistent with an aging theory which proposes that molecular damage to macromolecules leads to the structural and functional disorders associated with aging.

Screening for increased protein thiol oxidation in oxidatively stressed muscle tissue.

Elevated oxidative stress can alter the function of proteins through the reversible oxidation of the thiol groups of key cysteine residues. This study evaluated a method to scan for reversible protein thiol oxidation in tissue by measuring reduced and oxidized protein thiols. It assessed the responsiveness of protein thiols to oxidative stress in vivo using a dystrophic (mdx) mouse model and compared the changes to commonly used oxidative biomarkers. In mdx mice, protein thiol oxidation was significantly elevated in the diaphragm, gastrocnemius and quadriceps muscles. Neither malondialdehyde nor degree of glutathione oxidation was elevated in mdx muscles. Protein carbonyl content was elevated, but changes in protein carbonyl did not reflect changes in protein thiol oxidation. Collectively, these data indicate that where there is an interest in protein thiol oxidation as a mechanism to cause or exacerbate pathology, the direct measurement of protein thiols in tissue would be the most appropriate screening tool.

"Zero-length" cross-linking in solid state as an approach for analysis of protein-protein interactions.

We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum, and cross-links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H2(16)O and H(2)18O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides. We demonstrated specificity and reproducibility of our method by cross-linking homo-oligomeric protein complexes of glutathione-S-transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. The cross-linked peptides were validated against the GST monomer and dimer X-ray structures and by experimental (MS/MS) analyses. Some of the identified cross-links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze-drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy(3)2+ catalyzed, oxidative "zero-length" cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.

An image contrast agent selectively activated by prostate specific antigen.

A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents underwent activation both with enzymatically active prostate specific antigen (PSA) and alpha-chymotrypsin, releasing free fluorophore via cleavage of a three-component system. A hexapeptide derivative showed exclusive activation by PSA and constitutes a method for tracking PSA activity in vitro.

Synthesis and protein degradation capacity of photoactivated enediynes.

[structure: see text] The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics.

Capillary isoelectric focusing and affinity capillary electrophoresis approaches for the determination of binding constants for antibodies to the prion protein.

For the development of specific immunological assays, the binding of a specific antibody (Ab) to the target antigen (Ag) has to be relatively strong. In this study, we have utilized affinity capillary electrophoresis (ACE), a form of capillary zone electrophoresis (CZE) to determine the binding constant (Kb) of specific Abs against bovine serum albumin (BSA) and the healthy prion protein (PrPc), in buffer solutions at fixed pHs, approximating in vivo conditions. We have also utilized capillary isoelectric focusing (cIEF) to determine the complexity and recognition of the various isoforms of PrPc Abs towards their Ag, PrPc. Only ACE and CZE have been used to derive Kb values. The selected Abs for the prion protein can recognize both healthy and diseased states of the protein and are commercially available. The Kb values of PrPc Abs appear to be as strong as the anti-BSA (Ab to BSA) and other reported Kb values for proteins of similar size to PrPc. This appears to be one of the few reports on Kb values for any PrPc Abs, and their applications for in vitro immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)). Such assays are being used to detect the infectious agent, PrPres, in brain and related matter/tissues.

Image contrast agents activated by prostate specific antigen (PSA).

A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents underwent activation both with enzymatically active prostate specific antigen and alpha-chymotrypsin, releasing free fluorophore via cleavage of a three-component system.

Protein degradation with photoactivated enediyne-amino acid conjugates.

A series of photoactivated enediynes was prepared, and successfully employed for the selective degradation of target proteins.

Application of affinity capillary electrophoresis for the determination of binding and thermodynamic constants of enediynes with bovine serum albumin.

The binding constants and thermodynamic properties of a series of novel enediyne compounds with bovine serum albumin (BSA) were determined. The enediynes were synthesized, characterized, and then studied by affinity capillary electrophoresis (ACE) methods to derive these recognition parameters. Change in electrophoretic mobility of BSA as a function of enediyne concentration was determined at 25 degrees C providing binding constants of 1.76 x 10(5), 1.14 x 10(5), and 0.68 x 10(5) M(-1) for enediynephenylalanine carboxylic acid, enediynephenylalanine methyl ester, and enediyne carboxylic acid, respectively. The binding constant for the enediynephenylalanine carboxylic acid was in good agreement with that obtained using conventional methodology. Binding constants for the interaction of enediynes with BSA decreased with an increase in temperature. Van't Hoff plots showed a direct correlation between intensity of the binding constant and the sign and magnitude of various thermodynamic parameters (DeltaG, DeltaS, and/or DeltaH).

DNA cleavage by aromatic amines.

A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage, which may be ultimately applied to the development of sequence-specific agents.