Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay

摘要: A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biological source by carbonyl-DNPH hydrazone formation at acidic pH presence denaturing urea, and subsequent solubilization SDS stabilization acid hydrolysis 7.0. At this neutral (ntr) pH, interfering unreacted DNPH uncharged its thus increased hydrophobicity permits 100% effective removal solubilizate with ethyl acetate/hexane wash. The ntrDNPH more reliable sensitive than standard (std) because it eliminates main limitations: (i) (pKa 1.55) that nonspecifically bound to TCA 0.7)-protein pellet not effectively removed after wash EtOH: acetate positively charged, (ii) (TCA-induced) hydrazone, (iii) sample concentration re-determination, (iv) loss (TCA)-soluble proteins, (v) DNA interference, (vi) requires high quantity (≥ 1 mg). Considering assay's very low limit (1 µg), cumulative functional sensitivities are 2600- 2000-fold higher those stdDNPH assay, respectively. present study elucidates interference mechanism on also develops a standardized protocol treatment fractionation (into cytoplasmic/aqueous, membrane/lipid-bound, histone/DNA-bound proteins; see Supplement section V) order ensure reproducible carbonyl determination defined cell fractions, eliminate containing Cys sulfenic groups (via their neutralization dithiothreitol), streptomycin sulfate precipitation). Lastly, determines wall polysaccharides, paving way studies investigate walls acting as antioxidant defense plants, fungi, bacteria lichens.