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.     

A simple technique for eliminating interference by detergents in the Lowry method of protein determination.

The presence of nonionic and cationic detergents interfered in the Lowry method of protein estimation by causing precipitate formation. The addition of 0.5% sodium dodecylsulphate in the alkali reagent prevented this precipitation without affecting colour development, and allowed the method to be used on detergent treated membrane preparations.     

Lowry method of protein quantification: Evidence for photosensitivity

Despite reports of its susceptibility to various interfering factors, the Folin Phenol protein quantification method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265–275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken.     

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.     

A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates

Summary Clostridium thermocellum culture media containing crude cellulosic substrates, prepared by the steam-explosion of wood or straw, were found to interfere strongly with the Lowry method of protein determination. Investigations into the mode of interferences demonstrated that either the intercept (blank value) and/or the slope (intensity of color development) were affected. The efficiencies of precipitation procedures using the trichloroacetic acid method and the deoxycholate-trichloroacetic acid method were compared.     

Lowry determination of protein in the presence of sulfhydryl compounds or other reducing agents

The Lowry procedure has been modified for use in the presence of sulfhydryl compounds or other reducing agents by the addition of chloramine T under acid conditions before adding the alkaline-copper reagent. This method is applicable to the determination of 25–100 μg of protein, and a linear standard curve is obtained, though the sensitivity is slightly lower compared to that of the control.     

Lowry protein determination on membrane preparations: need for standardization by amino acid analysis

The protein content of three membrane protein preparations has been determined by the Lowry method with bovine serum albumin as a standard and also by quantitative amino acid analysis as an absolute method. The results differ considerably, the Lowry method giving 29–42% higher values. This implies that many published data for such proteins, based on Lowry protein determinations with bovine serum albumin as the generally applied standard, are in error. Suggestions are made on how to standardize the Lowry method so that reliable values can be obtained for membrane 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.     

Fiftyfold amplification of the Lowry protein assay

The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)