Protein Protocols Protein Protocols

32 Boerner et al.
Several approaches have been proposed to circumvent the aforementioned difficulties.
Protein precipitation with deoxycholate and trichloroacetic acid will eliminate
many interfering substances (10), but the inclusion of this step is laborious when multiple
samples are being analyzed simultaneously. Alternatively, samples can be lysed
in a buffer that is compatible with a particular assay, then diluted into a second buffer
after analysis. For example, cells lysed in Triton X-100 can be assayed for protein and
then diluted into SDS sample buffer in preparation for SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). This approach, however, can result in spurious results, as
illustrated by the demonstration that caspase-3 activation in lysates of mitogen-stimulated
lymphocytes reflects proteolytic cleavage occurring in the cell lysate rather than
in the cells prior to lysis (11,12). Finally, protein can be estimated by solubilizing
samples directly in SDS sample buffer, spotting them onto a solid support (e.g., glass
or nitrocellulose), washing away interfering substances, reacting the immobilized
polypeptides with Coomassie Blue, and eluting the Coomassie Blue to estimate the
protein content (13,14), but this process that is potentially time and labor intensive.
As a result of these shortcomings, there is still a need for alternative methods of
protein determination in biological samples. The nitric acid method reviewed in this
chapter represents one such newly devised method (15). Studies dating to the 1800s
have demonstrated that aromatic molecules undergo nitration when treated with nitric
acid. In particular, treatment of tyrosine with nitric acid produces 3-nitrotyrosine (16),
which is distinguished from the parent compound by the appearance of a new absorbance
peak at 358 nm. The method detailed in the following subheading utilizes this
chemistry to create a one-step method for protein determination in biological samples.
1.2. Nitric Acid Method for Protein Determination
1.2.1. Technique Derivation
Our laboratory has frequently examined the cellular accumulation of cytotoxic
platinum analogues (17,18). Because of concern about the efflux of platinum analogues
from cells during the course of manipulation (e.g., trypsinization and centrifugation),
we sought a method for the assessment of protein content after cells were washed in
situ and then solubilized directly in nitric acid, one of the matrices used for platinum
determination.
During the course of solublizing cells in nitric acid in preparation for platinum
determinations, we noted a brownish color change that appeared to vary in intensity
with the quantity of cells treated. Examination of the absorption spectrum of these
nitric acid lysates demonstrated a peak at 358 nm (Fig. 1A, inset) that was not present
in cells solubilized in SDS sample buffer or in nitric acid itself. The absorbance at
358 nm increased linearly with cell number; comparison of two human leukemia cell
lines, HL-60 (diameter 11 µm) and K562 (diameter 15 µm), revealed that the slope was
twofold higher in the larger cell line (Fig. 1A). We hypothesized that the new absorbance
peak at 358 nm reflected a reaction of nitric acid with one or more amino acids.
While nitric acid solubilization of L-tyrosine produced a sharp peak at 358 nm and
similar treatment of L-tryptophan produced a shoulder at the same wavelength, other
amino acids, including L-serine, L-arginine, L-phenylalanine, L-threonine, L-alanine,
L-proline, L-valine, L-histidine, L-methionine, L-isoleucine, L-glutamic acid, L-aspartic