Protein Protocols Protein Protocols

The Nitric Acid Method 31
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The Nitric Acid Method for Protein Estimation
in Biological Samples
Scott A. Boerner, Yean Kit Lee, Scott H. Kaufmann, and Keith C. Bible
1. Introduction
1.1. Background
The quantitation of protein in biological samples is of great importance and utility in
many research laboratories. Protein measurements are widely utilized to ensure equal
loading of samples on sodium dodecyl sulfide (SDS)-polyacrylamide gels and to provide
a basis for comparison of enzyme activities and other analytes.
Several methods are commonly employed for the determination of protein content
in biological samples, including the measurement of absorbance at 260 (1) or 205 nm
(2, and Chapter 1); the method of Lowry et al. (3, and Chapter 2), which relies on the
generation of a new chromophore on reaction of an alkaline protein hydrolysate with
phosphotungstic-phosphomolybdic acid in the presence of Cu2+ (4); the method of
Smith et al. (5, and Chapter 3), which relies upon the reduction of Cu2+ to Cu + , which
then forms a colored complex with bicinchoninic acid (BCA); the method of Bradford
(6, and Chapter 4), which relies on a change in absorbance on binding of Coomassie
Blue to basic and aromatic amino acids under acidic conditions (7); and the method of
Böhlen et al. (8), which relies on the reaction of fluorescamine with primary amines to
generate a fluorescent product. Unfortunately, compounds that are constituents of biological
buffers sometimes interfere with these methods, limiting their application.
Nucleic acids, neutral detergents of the polyoxyethylenephenol class, SDS, and urea
interfere with spectrophotometric determinations at 260 and 205 nm (9). Several
components commonly used in biological buffers, including tris-hydroxymethyl-amino
methane (Tris), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic [acid] (HEPES),
ethylenediaminetetraacetic acid (EDTA), neutral detergents, and reducing agents,
interfere with the method of Lowry et al. (10). In addition, Tris, ammonium sulfate,
EDTA, and reducing agents interfere with the BCA method, although neutral
detergents and SDS do not (5). Finally, many commonly used reagents, including
neutral detergents and SDS, interfere with the method of Bradford (6). Details of
compounds that interfere with these assays can be found in Table 1, Chapter 3.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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