A simple and sensitive detection of glutamic-pyruvic transaminase activity based on fluorescence quenching of bovine serum albumin

Article abstract of DOI:10.1039/c5ra24162f

It is well known that Cu(ii) can coordinate with l-alanine (Cu-Ala), which can be destroyed through the addition of glutamic-pyruvic transaminase (GPT) since GPT can effectively catalyze the conversion of l-alanine into keto-acetic acid. As a result, the free Cu(ii) ion can combine with bovine serum albumin (BSA) and in turn quench the fluorescence of BSA. In this context, a simple and sensitive GPT activity detection via fluorescence quenching method has been developed. The fluorescence intensity of the system shows a linear relationship with the GPT concentration in the range of 5 and 400 U L^-1 with a detection limit down to 3 U L^-1 (S/N = 3). Avoiding any labels or complicated operations, this cost-effective and convenient method holds the potential for the rapid diagnosis of GPT-related diseases.

Full text of DOI:10.1039/c5ra24162f

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A simple and sensitive detection of glutamic-
pyruvic transaminase activity based on
Cite this: RSC Adv., 2015, 5, 103557
fluorescence quenching of bovine serum albumin
a
a
ab
*
Miao Chen, Liya Rong and Xiaoqing Chen
It is well known that Cu(II) can coordinate with L-alanine (Cu–Ala), which can be destroyed through the
addition of glutamic-pyruvic transaminase (GPT) since GPT can effectively catalyze the conversion of L-
alanine into keto-acetic acid. As a result, the free Cu(II) ion can combine with bovine serum albumin
(BSA) and in turn quench the fluorescence of BSA. In this context, a simple and sensitive GPT activity
detection via fluorescence quenching method has been developed. The fluorescence intensity of the
system shows a linear relationship with the GPT concentration in the range of 5 and 400 U Lꢀ1 with
a detection limit down to 3 U Lꢀ1 (S/N ¼ 3). Avoiding any labels or complicated operations, this cost-
effective and convenient method holds the potential for the rapid diagnosis of GPT-related diseases.
Received 16th November 2015
Accepted 25th November 2015
DOI: 10.1039/c5ra24162f
www.rsc.org/advances
measuring based on a Y-shaped ow channel equipped with an
1. Introduction
on-chip L-glutamate sensor with the GPT activity up to
250 U Lꢀ1
.
Recently, Ruey-JenYang et al. developed a paper-
12
Glutamic-pyruvic transaminase (GPT; synonym alanine amino-
transferase, ALT), is an important amino transferase that can
catalyze the transfer of the amino group from L-alanine to
a-ketoglutarate.1 It is located in most cells in the body, but
found at the highest concentrations in the liver. Early studies
showed that GPT is a marker of liver disease due to the release
of GPT into the blood following liver cell necrosis.2 Being
through severe damage, GPT levels could rise up to 50 times
higher than the normal (about 400 U Lꢀ1).3,4 Therefore, GPT
activity detection is crucial to the diagnosis of diseases.
Conventional methods for GPT analysis mainly rely on colori-
metric, spectrophotometry and chromatography techniques.5
Generally, such techniques require relatively expensive
reagents, including enzymes and cofactors, and complicated
procedures along with the low accuracy.6–9 Consequently, in
order to address these problems, other alternative methods
were explored for GPT activity determination. For instance,
Rietz and Guilbault developed uorimetric methods to deter-
mine the activity of GPT both in solution and on a solid-
surface.10 Linear responses were observed in the ranges 5–106 U
Lꢀ1. I. Moser et al. established a miniaturized liquid handling
system comprising thin lm biosensor array for GPT determi-
nation.11 The system shows a linear relationship with the GPT
concentration in the range of 6 and 192 U Lꢀ1. Hiroaki Suzuki
based analytical devices for the determination of GPT activity
(0–125 U Lꢀ1).13 Despite these early encouraging efforts and the
extensive studies on the structure and mechanism of GPT,14,15
sensitive and rapid methods for GPT detection with wide
working range are highly desirable and urgent since the activi-
ties of GPT are being routinely determined in clinical labora-
tories, particularly for the diagnosis of liver diseases.
Bovine serum albumin (BSA), the most abundant protein in
the circulatory system, has been one of the most extensively
studied proteins.16–19 Containing tryptophan, tyrosine, and
phenylalanine residues, this protein possesses intrinsic uo-
rescence.20,21 It also has an affinity site with both histidyl and
carboxyl groups for Cu(II) ion. It has been reported that Cu(II)
ion could bind with BSA, leading to the uorescence quenching
of BSA.22 So the Cu(II)-catalyzed BSA uorescence quenching has
become one of the most effective methods for the detection of
Cu(II), which is characterized by low cost, mild conditions and
high sensitivity.23,24 A variety of compounds including dyes,
drugs, and quantum dots have been exploited for the selective
detection of Cu(II) based on the uorescence quenching of
BSA.25–29
It is worth mentioning that, in human circulatory system,
Cu(II) ions are migrated mainly in the form of complexes with
amino acids.30 The Cu(II)–amino acid complexes have attracted
enormous research attentions and interests due to their unique
structures and properties.31–33 Specically, Cu(II) complex with L-
alanine (Cu–Ala) is a mononuclear complex, and the central
Cu(II) ion is hexacoordinated with L-alanine.34,35 It has been re-
ported that the complexation between L-alanine and Cu(II) is far
stronger than that between BSA and Cu(II), which can prevent
et al. fabricated
a microuidic system for GPT activity
aCollege of Chemistry and Chemical Engineering, Central South University, Changsha
410083, China. E-mail: xqchen@csu.edu.cn; Fax: +86-731-88830833; Tel: +86-731-
88830833
bCollaborative Innovation Center of Resource-conserving
& Environment-friendly
Society and Ecological Civilization, Changsha, 410083, China
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the destruction of Cu–Ala by BSA.36–38 On the other hand, GPT
can catalyze the transfer of the amino group from L-alanine to a-
ketoglutarate and sabotage the complexation of Cu(II) and L-
alanine, resulting in the release of free Cu(II) ion.39–41 We
hypothesized that the subsequent combination of free Cu(II) ion
with BSA would lead to the uorescence intensity quenching of
the system, which should demonstrate a direct relationship
with the GPT activity. Herein, we report a sensitive and simple
technique for GPT activity detection employing the novel uo-
rometric quenching strategy.
3. Results and discussions
3.1. Principle of the detection system
The protocol of the proposed uorometric quenching method is
shown in Scheme 1. In the absence of GPT, since the
complexation between L-alanine and Cu(II) is far stronger than
that between BSA and Cu(II), it would be extremely difficult for
BSA to deprive Cu(II) from Cu–Ala complex. Therefore, only
a small amount of free Cu(II) ion that dissociated from Cu–Ala in
solution could bind with BSA and cause the binded BSA uo-
rescence quenching, the system still remains strong uores-
cence intensity. Through incubation with GPT, the
transamination of L-alanine caused the damage to the structure
of Cu–Ala, resulting in the release of more free Cu(II) ion.
Subsequently, the free Cu(II) ion can combine with BSA, which
results in larger degree quenching the uorescence intensity of
the system. As we know, the uorescence quenching directly
correlates with the concentration of GPT. It can be assumed that
GPT activity could be rapidly and precisely determined by virtue
of the uorescence quenching of BSA.
Preliminary experiments were carried out to verify our
presumption. As shown in Fig. 1, the GPT solution (curve a) and
the substrate solution (including a-ketoglutaric acid and PLP,
curve b) and Cu–Ala solution (curve i) showed very weak uo-
rescence signals, suggesting that GPT and Cu–Ala and the
substrate solution are non-uorescent and hardly interfere with
the assay. A very strong uorescence signal was observed for the
BSA solution (curve c). Aer incubation with GPT (curve d) and
substrate (curve e) respectively, strong uorescence signals were
still detected. Obviously, GPT and substrate were unable to
quench the intrinsic uorescence of BSA. As for the equivalent
Cu–Ala (200 mM, 20 mL, curve f) and Cu(II) (200 mM, 20 mL, curve
g) incubation with BSA respectively, the uorescence signal of the
former was much stronger than the latter. An explanation for this
phenomenon could be the amount of free Cu(II) ion in former
system was more than the latter system, more free Cu(II) ion
combine with BSA lead to more uorescence intensity quenching
of the system. It can be deduced that L-alanine can prevent the
binding of Cu(II) with BSA and only free Cu(II) ion in the system
can interact with BSA, in turn causing the uorescence
2. Experimental
2.1. Chemicals and reagents
Cupric nitrate (Cu(NO3)2$3H2O), L-alanine, pyridoxal phosphate
(PLP), a-ketoglutaric acid and other reagents were purchased
from Aladdin China Co. Ltd (Shanghai) and used as received.
Bovine serum albumin (BSA) was obtained from Sigma-Aldrich
Co. LLC. Glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) was
purchased from Sigma-Aldrich Co. LLC. One unit will convert
1.0 mmole of a-ketoglutarate to L-glutamate per min at pH 7.6 at
37 ꢁC in the presence of L-alanine. Cu–Ala was synthesized
according to the literature.37 All other chemicals were at least
analytical grade reagents and used directly without further
purication. Aqueous solutions were prepared with Milli-Q
water (18.4 MU cmꢀ1). Unless otherwise mentioned, all experi-
ments were carried out at room temperature.
2.2. Instruments
Fluorescence measurements were carried out on an F-7000
uorescence spectrometer (Hitachi Ltd, Shanghai, China)
using a square quartz absorption cells that can hold 4 mL of
solution. Excitation and emission slits were all set for a 10.0 nm
band-pass. The excitation wavelength was set at 280 nm, and
the emission spectra were collected from 290 to 470 nm. The
uorescence intensity at 340 nm was used to evaluate the
performances of the proposed assay strategy. UV-visible
absorption spectra were recorded using a Lambda 750 UV-vis
spectrophotometer. All measurements were carried out at
room temperature unless stated otherwise.
2.3. Procedure for GPT activity assay
GPT activity assay was performed according to the following
procedures. A 10 mL volume of GPT with a different nal
concentration ranging from 5 to 400 U Lꢀ1 was added to the
aqueous dispersion consisting of Cu–Ala (20 mL, 200 mM, pH ¼
7.0), a-ketoglutaric acid (10 mL, 200 mM) and PLP (10 mL, 5 mM)
at 30 ꢁC followed by incubation for 20 min. BSA (2 mL,
2 mg mLꢀ1) was added to the resulting mixture, which was then
incubated for 0.5 min. Subsequently, the data of uorescence
emission spectra were collected. The uorescence quenching of
BSA by Cu(II) ion was performed under the optimum conditions
described in earlier reports.22 Each point was detected for three
times, and the average values were used for quantication.
Scheme 1 Structure and sensing mechanism of fluorescent sensor for
GPT.
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the excess free Cu(II) ions will cause BSA protein denaturation.
Therefore, 200 mM Cu–Ala was used in the succeeding studies.
The inuence of the reaction time between Cu–Ala and GPT
was also examined. As shown in Fig. 2B, the uorescence
intensity at 430 nm weakened sharply with the increase of
reaction time between Cu–Ala and GPT, which tended to be
increased aer 20 min. The maximum uorescence quenching
was observed aer 20 min, which was chosen as the incubation
time. The effect of temperature on the system was also studied
since the temperature can affect the activity of GPT and the
stability of Cu–Ala.43 As illustrated in Fig. 2C, the uorescence
ꢁ
intensity of the system (without GPT) increased from 20 C to
50 ꢁC, which indicated that the stability of Cu–Ala increased (a).
ꢁ
At 30 C, the uorescence intensity of enzyme reaction system
(with GPT) reached the minimum (b), meaning that the activity
of GPT was the strongest at this point. At each temperature the
uorescence intensity of the system without GPT (a) was higher
than that of the system with GPT (b). The results indicated that
GPT initiate the enzymatic reaction and the transamination of L-
alanine caused the damage to the structure of Cu–Ala, resulting
in the release of more free Cu(II) ion. Subsequently results in
a larger degree decrease in uorescence intensity of the system.
Furthermore, the biggest difference of uorescence intensity
between (a) and (b) was obtained at 37 ꢁC. Although the activity
of GPT and the stability of Cu–Ala was not the largest at this
point, the uorescence quenching degree of the system caused
by GPT was largest. So the suitable temperature was found to be
37 ꢁC. Additionally, the pH values are also important to the
enzymatic processes and the stability of Cu–Ala. The results
(Fig. 2D) showed that the difference of uorescence intensity
between (a) and (b) was the biggest at pH 7.0. Therefore, pH
value was set at 7.0 for the following experiments.
Fig. 1 Fluorescence responses of the sensing system under different
conditions: (a) GPT solution (GPT: 10 mL 200 U Lꢀ1; deionized water:
2.04 mL). (b) Substrate solution (a-ketoglutaric acid: 10 mL 200 mM;
PLP: 10 mL 5 mM; deionized water: 2.03 mL). (c) BSA solution (BSA:
2.00 mL 2 mg mLꢀ1; deionized water: 50 mL). (d) GPT (10 mL 200 U Lꢀ1
;
deionized water: 40 mL) incubation with BSA (2.00 mL 2 mg mLꢀ1). (e)
Substrate (a-ketoglutaric acid: 10 mL 200 mM; PLP: 10 mL 5 mM;
deionized water: 30 mL) incubation with BSA (2.00 mL 2 mg mLꢀ1). (f)
Cu–Ala (20 mL 200 mM; deionized water: 30 mL) incubation with BSA
(2.00 mL 2 mg mLꢀ1). (g) Cu(II) (20 mL 200 mM; deionized water: 30 mL)
incubation with BSA (2.00 mL 2 mg mLꢀ1). (h) The mix solution con-
taining Cu–Ala (20 mL 200 mM), a-ketoglutaric acid (10 mL 200 mM),
PLP (10 mL 5 mM) and GPT (10 mL 200 U Lꢀ1) incubation with BSA (2.00
mL 2 mg mLꢀ1). (i) Cu–Ala solution (Cu–Ala: 20 mL 200 mM; deionized
water: 2.03 mL).
quenching. A uorescence quenching was clearly detected aer
the addition of GPT into the system (curve h) and the uores-
cence quenching degree was much stronger than the system
without GPT (curve f). This observation means that the enzymatic
reactions occurred and more free Cu(II) ions were formed.
Conclusively, GPT, Cu–Ala and substrate cause negligible effect
on the uorescence of the system and the release of free Cu(II) ion
from Cu–Ala catalyzed by GPT leads to BSA uorescence
quenching, which conrms the basic principle of our design.
3.3. Performance of the sensing system
The activity of GPT was tested by adding different activity units
of GPT into the test solution. Fig. 3A reveals the uorescence
spectra of the sensing system at different GPT concentrations.
The uorescence intensity decreased sharply with the
increasing of GPT concentration in the range of 5–400 U Lꢀ1
,
and decreased very slowly in the range of 400–500 U Lꢀ1. Fig. 3B
depicts the relationship between the uorescence intensity and
3.2. Optimization of the experimental conditions
It has been observed that GPT can catalyze the Cu–Ala to release GPT concentrations. In addition, a linear correlation (the linear
free Cu(II) and causing BSA uorescence quenching. If the equation is I ¼ 383.644 ꢀ 0.431CGPT, CGPT is the concentration
concentration of Cu–Ala is not high enough, Cu–Ala will be of GPT and I is the uorescence intensity, R2 ¼ 0.995) existed
quickly catalyzed by a small amount of GPT in the solution. The between the uorescence intensity and CGPT in the range of 5–
resulting narrow linear range won't comply with the analytical 400 U Lꢀ1 under the optimal conditions. The limit of detection
conditions of the whole enzymatic reaction. But if too much Cu– (LOD) was calculated according to the denition of 3sb/s, where
Ala is added, the excess free Cu(II) ions will cause BSA protein sb is the standard deviation of the blank samples and s presents
denaturation42 and then interfere with the corresponding uo- the slope.44,45 In this study, the uorescence signal of the blank
rescence tests. Thus, the inuence of the concentration of Cu– sample was measured for 10 times, and the standard deviation
Ala on the uorescence quenching degree were thoroughly (sb) was calculated. The LOD was nally determined to be
studied. As shown in Fig. 2A, the uorescence intensity 3 U Lꢀ1. It is worth noting that this obtained detection limit is
decreased sharply with the increasing of Cu–Ala concentration. clearly lower than that in the early report based on the colori-
And aer 200 mM it reached a plateau, this may attributed to metric ultramicromethod (10 U Lꢀ1).46 Moreover, the range of 5–
lack of Cu–Ala and GPT was excessive and all of the Cu–Ala was 400 U Lꢀ1 complies with the analytical conditions of the whole
transformed into free Cu(II) ions. But if more Cu–Ala was added, enzymatic reaction (Fig. 4).
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Fig. 2 (A) The concentration of Cu–Ala and the dependence of fluorescence quenching degree of the system. The concentration of GPT was
400 U Lꢀ1 (10 mL). (B) Effect of the reaction time between Cu–Ala and GPT on the fluorescence intensity of the system. (C) Effect temperature on
the fluorescence intensity of the system: (a), without GPT; (b), with GPT. (D) Effect of media pH on the fluorescence intensity of the system: (a),
without GPT; (b), with GPT.
glucosaccharase (Glu), alcohol dehydrogenase (ADH), xanthine
oxidase (XOD), and amylase were employed as the interferents
to investigate the selectivity of the proposed biosensor. The
concentrations of the used interferents were settled as TG 0.21
U,47 Glu 0.5 U,48 ADH 0.15 U,49 XOD 0.20 U,50 and amylase
3.4. Interference study
As we know, a large number of enzymes exist in the human
circulatory system, which might pose the potential interference
to the assay of GPT. Consequently, several representative
enzymes
including
glutamine
transaminage
(TG),
Fig. 3 (A) Fluorescence intensity of the system at different GPT concentrations. From (a) to (i): 0, 5, 25, 60, 100, 140, 200, 300 and 400 U Lꢀ1. (B)
Simulation curve between the fluorescence intensity and the GPT concentrations.
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4. Conclusions
In summary, a fast and sensitive method for GPT activity assay
based on the uorescence quenching of BSA was developed. It
has been found that GPT effectively destroyed Cu–Ala complex
to release free Cu(II) ion, which subsequently combined with
BSA leading to the uorescence quenching. Various parameters
of this proposed method has been extensively optimized. The
superior performance of this technique including low LOD (3 U
Lꢀ1), wide working range (5–400 U Lꢀ1) and high selectivity
would pave the way for ultimately realizing the rapid and
simplied diagnosis of GPT or GPT-related diseases.
Conflict of interest
The authors have declared no conict of interest.
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Acknowledgements
We gratefully acknowledge the nancial support from National
Natural Science Foundation of China (21175155, 21475152),
and the Specialized Research Fund for the Doctoral Program of
Higher Education of China (20130162110017).
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