Protein Protocols Protein Protocols

The Nitric Acid Method 33
Fig. 1. Treatment of cells or L-tyrosine with nitric acid results in an increase in absorbance at
358 nm. (A) Relationship between cell number and absorbance at 358 nm in HL-60 and K562
cells solubilized in nitric acid for 24 h. Inset, absorption spectrum resulting from treatment
of OV202 cells with nitric acid. (B) Absorption spectra resulting from treatment of L-tyrosine,
L-tryptophan, L-serine, or L-arginine with nitric acid for 1 h. Amino acid concentrations in nitric
acid were all 0.1 mg/mL.
acid and L-asparagine failed to yield products that absorbed at 358 nm (Fig. 1B and
data not shown). Nucleotides (ATP, CTP, GTP, TTP, UTP), RNA, and DNA also failed
to yield products that absorbed at 358 nm. These investigations led us to propose that
the 358 nm absorption peak observed after nitric acid solubilization of cells likely
primarily reflects the tyrosine content of cellular proteins.
To understand better the chemistry responsible for these observations, we solublized
L-tyrosine in nitric acid for 1 h. A single reaction product that co-migrated with authentic
3-nitrotyrosine by high performance liquid chromatography (HPLC) and had an
absorbance maximum at 358 nm was formed (Fig. 2). Incubation for prolonged periods
of time resulted in disappearance of the 3-nitrotyrosine peak and appearance of
additional peaks that are thought to reflect nitration at additional sites as well as oxidation
(data not shown). As a consequence, the absorbance at 358 nm reached a peak
between 1 and 6 h, and then subsequently declined.
To determine whether these observations could be utilized to devise a method for
quantitating protein, bovine serum albumin (BSA) was solubilized in nitric acid and
incubated for varying lengths of time. As indicated in Fig. 3A, absorbance at 358 nm
measured after incubation for 0.5–70 h was a linear function of protein content. Examination
of these data revealed that production of the chromophore was approx 88%
complete within 30 min, reached a maximum at approx 24 h, and remained stable thereafter
(Fig. 3A, inset). We hypothesize that the stability of the 358 nm peak in proteins
treated for prolonged periods with nitric acid (in contrast to the disappearance of the
same peak when L-tyrosine is subjected to prolonged nitric acid treatment) results from
diminished susceptibility of tyrosyl residues in protein to reaction beyond 3-nitration.
To determine whether this approach could be utilized to quantitate protein under
conditions compatible with SDS-PAGE, aliquots of BSA in SDS sample buffer