Lowry determination of protein in the presence of Triton X-100.

The Lowry procedure has been modified for use in the presence of Triton X-100 (TX-100) by the addition of 10% sodium dodecyl sulfate. The method is applicable to samples containing 40–120 μg protein.     

Assay of protein in the presence of high concentrations of sulfhydryl compounds

A simple procedure is described for removal of Triton X-100 from protein samples by adsorption of the detergent on a commercial copolymer in bead form. The concentration of detergent can be lowered to approximately 0.01% with no loss or dilution of protein.     

Interference by detergents, chelating agents, and buffers with the Lowry protein determination

EDTA distorts the Lowry method even at the concentration of 0.5 mm. The effect of EDTA, glycine, glycylglycine, Tricine, and Tris on the Lowry method can be calibrated only at a fixed concentration of the chemicals. The effect of succinic acid, sodium citrate, and Bicine on the Lowry method changes depending on the concentration of the chemicals, but the changes were not as significant as in the case of the chemicals mentioned above. Sodium phosphate at pH 8.9, sodium dodecyl sulfate, sucrose, and urea do not affect the Lowry method when a reference containing only the chemical is used.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Artificial reductant enhancement of the Lowry method for protein determination

Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used.     

A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents

A method is described for quantitation of protein in the presence of reducing agents, detergents, and other substances which often interfere with assays of protein in solution. The proteins are applied to Whatman No. 1 filter paper, air-dried, washed with methanol, and then stained with Coomassie brilliant blue G. Following destaining, the paper is air-dried and the protein-bound dye is extracted. Sample absorbance measurements are made in a 96-well plate using an automated microplate reader (600-405 nm) or in a cuvette at 610 nm. This filter paper assay is useful for determining 100 ng to 20 micrograms of protein in the presence of ammonium sulfate, urea, thiol-reducing agents, amino acids, DNA, ionic and nonionic detergents, and acid or base.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

An improved colorimetric method for the determination of proline in the presence of other ninhydrin-positive compounds

1. The conditions required for sensitive and specific colorimetric determination of proline with acidified ninhydrin were investigated. 2. A method applicable to protein samples was developed. 3. The only compound found to interfere appreciably was a hydroxyproline.     

Hemoprotein-mediated reduction of nitrated DNA bases in the presence of reducing agents

DNA damages by reactive nitrogen oxide species may contribute to the multistage carcinogenesis processes associated with chronic infections and inflammation. The nitrated DNA adducts 8-nitroguanine (8NG) and 8-nitroxanthine (8NX) have been shown to derive from these reactive nitrogen oxide species, but they are not stable in DNA since they undergo spontaneous depurination. We herein report that hemin and hemoproteins, including hemoglobin and cytochrome c, mediate reduction of 8NG and 8NX to their corresponding amino analogues in the presence of reducing agents under physiologically relevant conditions. This reaction is believed to involve the reduced heme moiety produced from the reduction of oxidized hemoglobin or cytochrome c by reducing agents. The combination of hemoglobin and dihydrolipoic acid generated the reduced products in high yields. Ascorbate, quercetin, and glutathione are also capable of reducing these nitrated DNA adducts. The hemoglobin macromolecule reduces 8NG and 8NX formed in nitryl chloride-treated calf thymus DNA, as evidenced by the formation of the amino adducts using reversed-phase HPLC with photodiode array detection. Hemin is more efficient than equal molar of heme on hemoglobin in reducing 8NG-containing DNA, indicating the role of protein in impeding the reaction. Furthermore, we also show that the reduction product 8-aminoguanine is persistent on DNA. These findings suggest that reduction of nitrated DNA by the heme/antioxidant system might represent a possible in vivo pathway to modify DNA nitration.