2-4-dinitrophenylhydrazine and sodium-borohydride

Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.

Topics: Biochemistry; Biotin; Borohydrides; Cysteine; Isoaspartic Acid; Isotope Labeling; Oxidation-Reduction; Phenylhydrazines; Protein Binding; Proteins; Sulfhydryl Compounds; Tyrosine

Protein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.

Topics: Biochemistry; Borohydrides; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Iodoacetamide; Isoaspartic Acid; Mass Spectrometry; Oxidation-Reduction; Phenylhydrazines; Protein Carbonylation; Proteins; Reference Standards; Spectrophotometry; Sulfhydryl Compounds; Tritium; Tyrosine

Nonreductive modification of proteins with glyceraldehyde forming 2-oxo-3-hydroxypropylated protein is mechanistically analogous to nonenzymic glycation reactions. The latent cross-linking potential of glyceraldehyde as a consequence of the reactivity of the carbonyl function of 2-oxo-3-hydroxypropyl groups of nonreductively modified protein has been now investigated. Reaction of RNase A (0.5 mM) with glyceraldehyde (20 mM) at pH 7.4 and 37 degrees C for 4 h resulted in the intermolecular cross-linking of the protein, with the concomitant development of a yellow chromophore with two new absorption bands having maxima around 305 and 375 nm. The product exhibited a fluorescence that had excitation and emission maxima around 365 and 450 nm, respectively. The presence of NaCNBH3 during the reaction, which selectively reduces the Schiff base adducts of aldotriose to form 2,3-dihydroxypropyl groups on proteins, inhibited both the cross-linking reaction and the development of the absorption and fluorescence characteristics. The hydroxymethyl group of the aldotriose is not an essential moiety since the cross-linking potential of glyceraldehyde is comparable to that of glyceraldehyde 3-phosphate. The formation of cross-links appears to involve the carbonyl function of the keto amines resulting in the formation of Schiff base adducts (ketimine linkages) as the initial event. Consistent with this, incubation of 2-oxo-3-hydroxypropylated RNAse A with [14C]glycine ethyl ester resulted in the incorporation of the reagent into the protein. The cross-linking reaction was inhibited when the reaction of RNase A with glyceraldehyde was carried out in the presence of amino compounds, such as glycine ethyl ester, ethanolamine, glucosamine, and aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amines; Borohydrides; Cross-Linking Reagents; Glyceraldehyde; Guanidines; Ketones; Oxidation-Reduction; Phenylhydrazines; Proteins; Ribonuclease, Pancreatic; Spectrometry, Fluorescence