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pH of normal cells falls in the range of 7.2–7.5 which sharply
reduces in tumor cells to around 6.7–7.0 which is believed to
be a result of abnormal glycolysis in these cells.[27,28] Paquette
et al. reported that the activity of NO frequently increases in
breast cancer cells and oxidation of estrogen (which stimulates
breast cancer cells) often leads to the production of peroxyni-
trite.[29] NT has been detected in significant amounts in lymph
node metastasis, in breast cancer, and immune staining has
shown that NT is present in significant amounts within inflam-
matory cells located at the sides of the blood vessels at the
edges of tumor cells.[30,31] Nitration of Tyr is sensitive to pH and
pH profile studies have been carried out during the formation
of NT. Cytochrome c mediated tyrosine nitration in presence of
hydrogen peroxide showed a higher level of NT production at
pH 5, that is, in the acidic region.[32] Carbonate mediated nitra-
tion in phenolic compounds over a range of pH 4–8.5 showed
that the maximum nitration effect is observed at pH 6.5.[33]
Effect of tyrosine nitration on spermine NONOate showed in-
teresting results where the NT formation reached maximum
enzymatic activity of proteins like RNase T1[48] during in vitro
experiments. Chain length in PEG molecules often play a cru-
cial role as seen from PEG-protein interaction studies where
the association constant values for PEG 10 K and PEG 200 K
with bovine serum albumin (BSA) are reported to be 2.55ꢀ
105 mꢀ1 and 3.42 ꢀ105 mꢀ1 respectively.[49]
In the present study, we have investigated the effect of per-
oxynitrite (PN) at pH 7.4 and 6.0 on RNase A in PEG 400 and
PEG 6000 respectively and compared the degree of nitration in
each case. In addition, we have also studied the secondary
structure of RNase A upon NT formation and investigated how
the nitration process affects the ribonucleolytic activity of
RNase A. Fluorescence lifetime measurements have been per-
formed to predict the Tyr residue(s) which are likely to under-
go nitration.
2 Results and Discussion
C
levels in presence of gaseous NO at pH 8.2, whereas when
The objective of this study is to compare the degree of Tyr ni-
tration at pH 7.4 and pH 6.0 and investigate how crowding
agents (PEG) of different chain lengths (PEG 400 and PEG
6000) can affect the process of protein nitration. The effect of
nitration on the structure and enzymatic activity has also been
studied with the help of FTIR and UV based enzyme kinetics
assays, and the specific Tyr residues undergoing nitration have
been monitored using fluorescence lifetime studies. RNase A is
an endoribonuclease whose primary function is to catalytically
cleave RNA during protein synthesis and proper functioning of
this enzyme is essential for maintaining an optimum balance
during protein synthesis. Oxidative stress in mammalian cells
can lead to the excess production of the superoxide radical
which reacts with nitric oxide (one of the major signaling mol-
ecules in neurons) to yield peroxynitrite (PN).[37,50] PN mediated
nitration of Tyr residues is one of the well-known posttransla-
tional protein modifications which are found to affect the
structure and activity of proteins and it is also associated with
pathological processes.[51] RNase A has six Tyr residues and no
Trp residue, so it possible to monitor nitrotyrosine formation
spectrophotometrically without Trp interference.[52] Nitration
studies of RNase A reported herein investigate how nitrotyro-
sine formation can alter the enzymatic activity of the protein
and how a crowded medium can affect the overall process of
nitration.
peroxynitrite solution was used, the NT formation reached a
maximum at pH 7.4.[17,34] A similar trend was also observed in
SOD and Fe3+ EDTA catalyzed nitration of 4-hydroxyphenylace-
tic acid (4-HPA) which showed maximum nitration at pH 7.5
and it has also been found to fall sharply with further elevation
in pH.[35] The variation in the degree of nitration has a strong
pH dependence on the peroxynitrite species, which forms the
basic source for tyrosine nitration.
Ribonuclease A (RNase A) is a basic protein (pI ꢁ9.3) with
six Tyr residues (at positions 25, 73, 76, 92, 97 and 115) and the
active site contains His12, Lys41 and His119 residues which are
involved in the catalytic cleaving of RNA during the transcrip-
tion and translation steps of protein syntheses.[36] Previous re-
ports on nitration of RNase A using tetranitromethane showed
that a maximum of three Tyr residues out of six can be nitrated
and a later HPLC based analysis showed that Tyr115 and Tyr76
are nitrated to the maximum extent with a certain degree of
nitration at Tyr73 but at very high concentrations of peroxyni-
trite.[37,38] Vaz et al. compared the action of myeloperoxidase
(MPO) and PN on Ribonuclease A and showed that MPO in
presence of hydrogen peroxide and nitrite can nitrate Tyr115
whereas PN is much less specific and may nitrate almost all
the Tyr residues.[39] However, these studies were carried in
aqueous solution in presence of buffers which are dilute in
comparison to the cellular medium. Around 40% of cells are
occupied by various solutes or macromolecules thereby
making biological systems more concentrated.[40,41] Proteins
and RNA respectively occupy up to 200–300 gLꢀ1 and 75–
150 gLꢀ1 of the cellular medium whereas oligosaccharides and
other solutes make up ꢁ30% of the cytoplasm.[42,43] Molecular
crowding agents which consist of polysaccharides like Dextran
and Ficoll or polyethers like poly-ethylene glycol (PEG), can
mimic the cellular environment.[44,45] Earlier reports showed
that PEG can promote nitration in aromatic carboxylic acids de-
pending upon the molecular weight.[46] Apart from mimicking
the cellular environment, these compounds increase the stabil-
ity of proteins like the chymotrypsin inhibitor 2[47] as well as
2.1 Characterization of peroxynitrite
The UV/Vis spectra of peroxynitrite show a characteristic ab-
sorption peak around 300 nm. (Figure 1A) For absorption
measurements, the stock solution was diluted to different con-
centrations with 600 mL of 100 mm NaOH. No peak shift is de-
tected in the absorption spectra with varying concentrations
of peroxynitrite. The concentration of peroxynitrite (PN) was
calculated using the Beer–Lambert law from the absorbance
value at 302 nm[53] and reported molar absorption coefficient
value[54] at this wavelength of 1670mꢀ1 cmꢀ1
.
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