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KOWACZ AND WARSZYŃSKI
a prerequisite for operative catalytic process. It has been recognized
that nitrogen from the guanidine group of arginine (Arg410) serves
as a proton accepting site, but participation of lysine (Lys414) and his-
tidine (His242) in proton transfer reaction on hydrolysis of esters has
also been proposed.2 It is recognized that directed proton transfer
can be supported by formation of water wires connecting donor and
acceptor sites.5 Furthermore, there are studies indicating that struc-
tural correlations of water molecules can be enhanced by electromag-
netic radiation, especially in the infrared range.6,7 After transient
acetylation of albumin by nucleophilic attack of tyrosine on the sub-
strate, the deacetylation step takes place by reaction with water mol-
ecule. Hydrolysis of nitrophenyl esters in particular leads to formation
of nitrophenol.1
absorption band centered around 400 nm was monitored with the
UV–visible (Vis) spectrophotometer (the UV‐1800 from Shimadzu) to
follow conversion of p‐NPA to p‐NP. The evolution of UV–Vis spectra
was recorded until they have fully developed and no further changes
could be observed (usual reaction time was 5 min.). Presented in this
work are those final spectra for the completed reaction. Control
experiments, accounting for spontaneous hydrolysis of p‐NPA, were
performed in the same manner but with buffer solution instead of pro-
tein in the reaction media.
In order to access a potential effect of infrared radiation (IR) on
BSA‐supported transformation of p‐NPA, the protein solution was
exposed to IR light prior to its mixing with the substrate. After the
10‐minute exposure, all experimental procedures were exactly the
same as described above. Light‐emitting diode with the emission max-
imum in the IR range at wavelength λ = 2900 nm (LED29, Roithner‐
Lasertechnik) and the full width at half maximum of 350 nm, operated
by D‐31M driver (Roithner‐Lasertechnik) in a quasi‐continuous wave
mode (the mode of maximum average optical power from the LED)
at 2 kHz and 200 mA was used as the source of IR light. The emitted
IR light represents a non‐ionizing electromagnetic radiation (strongly
absorbed by liquid water) and its maximum optical power ranged to
29.47 mW. During irradiation, samples were thermostated at 22°C
with the use of Echotherm IC20 dry bath (Torrey Pines Scientific),
and their temperature was constantly monitored with TW2 micro-
probe thermometer (ThermoWorks) by inserted micro thermocouple.
To address effect of temperature, additional experiments were per-
formed with samples equilibrated at 40°C before contacting the
reactants.
p‐nitrophenol (p‐NP) is a widespread environmental pollutant,
toxic to humans and animals. It can cause damage to the central ner-
vous system, liver, or kidney. Repeated exposure might result in blood
cells injury and mutagenic effects. Therefore, the effective ways of
degradation of p‐NP are still sought and extensively studied. There
are two major metabolic oxidation pathways proceeding by removal
of nitro group by specific enzymes—monooxygenases and formation
of either hydroquinone or benzenetriol as the terminal aromatic inter-
mediates that undergo ring cleavage.8 Apart from biological conver-
sion, p‐NP can be oxidized via chemical treatment with oxidizing
agents such as hydrogen peroxide or ozone among others. Anaerobic
biological degradation or catalytic reduction (eg, with the use of nano-
particles) leads in general to formation of 4‐aminophenol.9-11 Aqueous
photolysis of nitrophenols by solar ultraviolet (UV) radiation proceed-
ing via radical formation results in release of nitro group in the form of
nitrous acid.12
For particular experiments, samples of protein solution were ini-
tially degassed with the use of vacuum pump (Buchi, V‐100) set at
50 mbar for 1 hour. Samples were then exposed to IR in sealed
cuvettes from IR transparent quartz.
In this study, we explore the activity of BSA toward p‐NPA to
show new routes of this interaction going beyond usually considered
hydrolysis step and including protein‐supported transformation of
p‐NP. We also demonstrate the potential of the remote physical trig-
ger—infrared radiation to affect those chemical transformations.
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3
RESULTS AND DISCUSSION
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As a result of esterase‐like activity of BSA p‐NPA is converted to p‐NP
(Scheme 1.). The transformation is usually evidenced by monitoring
evolution of the UV–Vis band centered around 400 nm, which corre-
sponds to the absorption of nitrophenolate ion13 (yellow at pH values
above its pKa = 7.08 at 22°C).
2
EXPERIMENTAL SECTION
Stock solution of BSA (from Sigma, 94158) was prepared in in 20‐mM
imidazole buffer of pH = 7.4 or for supplementary experiments in
20‐mM Tris (tris [hydroxymethyl]aminomethane) buffered saline of
pH = 7.4. Choice of the imidazolium buffer was based on previous
experimental data obtained by the authors on the behavior of BSA
in this media including structural response of the protein to infrared
light. Furthermore, in its physiological environment, serum albumin
molecule is exposed to imidazole groups of other proteins exerting
also their buffering action.
In this study, we have observed that with increasing concentra-
tion of BSA in the reaction mixture, the band position is significantly
shifted toward lower wavelengths as shown in Figure 1.
p‐nitrophenyl acetate (p‐NPA from Sigma, N 8130) was dissolved
in methanol (Uvasol for spectroscopy, 106002 from Merk) and stored
refrigerated. Immediately, before using, the p‐NPA solution was
diluted with Milli‐Q water (1:100 proportion) to get working solution
of concentration of 3.5 mM. In order to verify esterase‐like activity
of BSA, 100 μL of 3‐mM solution of p‐NPA was added to 400 μL of
the protein solution of 3‐μM concentration (unless specified other-
wise for particular experiments). Subsequently, the evolution of the
SCHEME 1 Bovine serum albumin (BSA)‐assisted hydrolysis of p‐
nitrophenyl acetate