Interaction of mercury and copper on papain and their combined inhibitive determination

Article abstract of DOI:10.1016/j.bej.2015.01.001

Influence and interaction of mercury ion (Hg²⁺) and copper ion (Cu²⁺) on papain activity in casein hydrolysis were investigated. Single Hg²⁺ or Cu²⁺ at low concentrations induced an increase in papain activity, but decreased it at high concentrations, confirming a typical hormesis phenomenon. The interaction of Hg²⁺ and Cu²⁺ at various concentration combinations showed that the binary interaction of 10^-8mol/L Cu²⁺ and 10^-6mol/L Hg²⁺ (Binary union S) buffer was of synergistic nature, while 10^-4mol/L Cu²⁺ and 10^-4mol/L Hg²⁺ (Binary union I) buffer was of competitive inhibition. The conformational changes in papain structure due to the interaction of binary metal ions were studied by ATR-FTIR, UV-vis and intrinsic fluorescence spectroscopies, also the changes of papain catalytic behavior were studied through kinetic analysis. Decreasing of α-helix content with increasing in intermolecular β-sheet aggregates content in Binary union I buffer resulted in an inactivation of papain activity by 57.2% and lower affinity for casein. On the contrary, papain activity increased with α-helix content increasing and intermolecular β-sheet aggregates content decreasing in Binary union S buffer. The competitive interaction between Cu²⁺ and Hg²⁺ on papain activity was found at higher concentrations (≥10^-4mol/L), and the inhibition of the binary metal ions on papain was of a noncompetitive type.

Full text of DOI:10.1016/j.bej.2015.01.001

Biochemical Engineering Journal 97 (2015) 125–131
Contents lists available at ScienceDirect
Biochemical Engineering Journal
journal homepage: www.elsevier.com/locate/bej
Regular article
Interaction of mercury and copper on papain and their combined
inhibitive determination
Xue-Ying Liua,b, Hong-Yan Zeng , Meng-Chen Liao , Bo Feng
Bi Foua Claude Alain Gohi
a
a,∗
a
a,∗
,
a
Biotechnology Institute, College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan, China
Hunan Key Laboratory of Green Packaging and Application of Biological Nanotechnology, Hunan University of Technology, Zhuzhou 412007, China
b
a r t i c l e i n f o
a b s t r a c t
Influence and interaction of mercury ion (Hg2+) and copper ion (Cu2+) on papain activity in casein hydrol-
Article history:
Received 14 October 2014
Received in revised form
2+
2+
ysis were investigated. Single Hg or Cu at low concentrations induced an increase in papain activity,
but decreased it at high concentrations, confirming a typical hormesis phenomenon. The interaction of
1
9 December 2014
2+
2+
−8
2+
Hg and Cu at various concentration combinations showed that the binary interaction of 10 mol/L
Accepted 7 January 2015
Available online 8 January 2015
2
+
−6
2+
−4
Cu and 10 mol/L Hg (Binary union S) buffer was of synergistic nature, while 10 mol/L Cu and
−4
2+
1
0
mol/L Hg (Binary union I) buffer was of competitive inhibition. The conformational changes in
papain structure due to the interaction of binary metal ions were studied by ATR-FTIR, UV–vis and intrinsic
fluorescence spectroscopies, also the changes of papain catalytic behavior were studied through kinetic
analysis. Decreasing of ␣-helix content with increasing in intermolecular ␤-sheet aggregates content in
Binary union I buffer resulted in an inactivation of papain activity by 57.2% and lower affinity for casein.
On the contrary, papain activity increased with ␣-helix content increasing and intermolecular ␤-sheet
Keywords:
Protease
Biocatalysis
Enzyme activity
Mercury ion
Copper ion
2+
aggregates content decreasing in Binary union S buffer. The competitive interaction between Cu and
Kinetic parameters
2+
−4
Hg on papain activity was found at higher concentrations (≥10 mol/L), and the inhibition of the binary
metal ions on papain was of a noncompetitive type.
©
2015 Published by Elsevier B.V.
1
. Introduction
aforementioned studies were mostly concentrated on the effect of
a single heavy metal ion. However, contaminated systems usually
contain various heavy metals rather than a single one in the real
environment. As a consequence, enzymes are exposed to multi-
ple metal ions system. Still, most enzyme biosensors detect heavy
metals in contaminated water and derive single-substance toxicity
data from single-substance criteria, but neglecting the interaction
effects among the mixture. So, the detecting methods for the pres-
ence of two or more ions are thus urgently needed [5].
Papain is a highly stable enzyme, one of the proteolytic enzymes
of papaya latex [16]. Papain has been widely used as biosensors for
its wide pH span for optimum activity, high sensitivity, tempera-
ture stability, low price, and short response time. But few influence
assays on mixed heavy metal ions based on papain have been
Due to incessant industrial development, heavy metal con-
taminants have been introduced into water environments and
become an increasingly serious social problem. Owing to their bio-
accumulation and non-degradability, heavy metals pose a serious
pollution hazard to the aqueous environment [3,4,7,20]. Certain
metal ions are highly toxic, and the determination of trace toxic
heavy metals in water environments has become highly impor-
tant. Among all of them, Hg2+ has attracted the most attention
due to its strong toxicity and increasing level of its extended
use in industrial processes [12]. In order to evaluate the toxic-
ity, bioavailability, bioaccumulation and transport of the heavy
metal elements more readily, sensitive analytical procedures are
required for detection [2]. Numerous enzymes have been utilized
for heavy metal detection, for enzyme activity is an indicator of
the toxicity of heavy metals and other pollutants [1,11,15,22]. The
2
+
reported, particularly on inhibition [20]. Metal ions such as Cu
,
2+
2+
2+
Hg , Zn , and Pd were found not only binding to papain but
also inhibiting its activity partly or completely. The IC of Hg2+
,
50
2+
2+
2+
Ag , Pb and Zn were 0.39, 0.40, 2.16 and 2.11 mg/L, respec-
tively, while for Cu and Cd , the limit of quantitation were 0.004
and 0.1 mg/L, respectively [20].
2+
2+
∗ Corresponding authors. Tel.: +86 731 58298175; fax: +86 731 58298167.
E-mail addresses: hongyanzeng99@hotmail.com (H.-Y. Zeng),
fengbo@xtu.edu.cn (B. Feng).
In the preliminary stage of our work, we observed that the inter-
active effect of the two mixed metal ions on papain activity was
http://dx.doi.org/10.1016/j.bej.2015.01.001
1
369-703X/© 2015 Published by Elsevier B.V.

1
26
X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
different from that of single Hg2+ and Cu2+, while the inactiva-
2+
tion of papain activity was about 58% for Cu and nearly 100%
2+
for Hg . The inactivation mechanism affected by the binary ions
2
+
2+
(
Hg & Cu ) was still not clearly understood. The principal aim of
2+
this present work was to evaluate the combined effect of Hg and
2
+
Cu on papain activity and structure. The ATR-FTIR, UV–vis intrin-
sic fluorescence spectroscopies and kinetics analysis were used to
investigate the structure-function relationship in the presence of
the binary ions.
Scheme 1. The irreversible reaction mechanism of the binary metal ions.
The papain (0.5 mg/mL) was equilibrated in the solutions with
− −6
8
2+
2+
2
. Materials and methods
the binary ions of 10 mol/L Cu + 10 mol/L Hg (Binary union
−
4
2+
−4
2+
S) and 10 mol/L Cu + 10 mol/L Hg (Binary union I) under
◦
2.1. Enzyme and regents
25 C for 10 min, respectively, and then centrifuged at 3000 rpm
equal to g value 800) for 4 min. The papain without any metal ions
(
Papain (EC3.4.22.2, ≥99%), bovine serum albumin (BSA), tyro-
treated was used as the control. The supernatant was used for ATR-
FTIR, UV–vis and fluorescence spectral measurements. Triplicate
samples were analyzed and the data obtained from the triplicate
runs were averaged and used as the final result.
sine and casein were purchased from Sigma–Aldrich Company Ltd.
All other reagents used were of analytical grade and used without
further purification. All solutions were prepared with redistilled
and ion-free water.
2.5. Kinetic measurements
2.2. Effect of single metal ion on papain activity
The kinetic model of substrate reaction during irreversible mod-
Papain solution (1.0 mg/mL) was obtained by dissolving the
ification of enzyme activity described by Zhao and Tsou was used
to study the kinetics of casein hydrolysis by papain with the
binary metal ions [24]. The reaction mechanism was considered
in Scheme 1, where E, S, P and Y represent papain, substrate casein,
product tyrosine and the binary ions, respectively. EY, ES and EYS
were the respective complexes.
enzyme in Tris–HCl buffer (0.1 mol/L, pH 7.0). Stock solution of
HgCl2 (0.1 mol/L) and CuCl2 (0.1 mol/L) were prepared in the
Tris–HCl buffer and diluted into the concentrations varied from
−
10
−2
1
0
to 10 mol/L for papain activity assays. Firstly, papain
solution (1 mL) was added into the buffer (1 mL) of different
◦
As was usual the case, [S] » [E ] and that the modification
metal ion concentrations at 40 C, secondly casein solution (3.0 mL,
0
2
0.0 mg/mL) was added into the mixture 10 min later. The reac-
reactions were relatively slow compared with the setup of the
steady-state of the enzymatic reaction. The product formation can
be written as:
◦
tion was carried out at 40 C for 30 min and then stopped by 2.0 mL
trichloroacetic acid (TCA). The activity of papain was determined by
a Hitachi U-2001 spectrophotometer at 275 nm. One unit of enzyme
activity (U) was defined as 1 ␮g tyrosine formed per minute at 40 C
and pH 7.0. The relative activity (%) was the ratio of the enzyme
ꢀ
v − v
ꢀ
(1 − e−At
◦
[P]t = v t +
)
(1)
(2)
A
ꢀ
k Km + k [S]
2+
2+
0
0
activity in the Tris–HCl buffer with different Hg or Cu concen-
trations to the corresponding enzyme activity without Hg or Cu
A =
Km + [S]
2+
2+
.
All the experiments were carried out at least three experiments in
each experimental group and the average number was employed
as the statistical analysis indicator.
where [P]t was the concentration of the product formed at time
t. A was the apparent rate constants. [S] was the concentration
of casein. v and v were the reaction velocities of reaction in the
ꢀ
absence and presence of the binary ions at time t, respectively. Km
ꢀ
ꢀ
and Km were the Michaelis constants. k and k were the dissocia-
2
.3. Effect of the binary ions on papain activity
0
0
tion constants for the modifier with different forms of the enzyme,
respectively. Vm and Vm were maximum reaction velocities. When
v> v , the binary metal ions modifier was an activator. When v< v ,
the modifier binary ions was an inhibitor. When t was sufficiently
long, the curves become straight lines and the product concentra-
tion was written as [Pe]:
ꢀ
In order to determine the interactive effect of Hg2+ and Cu2+
ꢀ
ꢀ
ions on papain activity, the assay was performed by incubating the
papain in the binary ions buffer. The buffer was prepared by mixing
equal volume of Hg and Cu buffers at different concentration.
Papain activity in the presence of the mixed metal ions was also
monitored as described above.
2
+
2+
ꢀ
1
Pe]
k · Km
1
[S]
k0
0
=
·
+
(3)
[
Vm
Vm
2.4. ATR-FTIR, UV–vis and intrinsic fluorescence spectroscopies
3. Results and discussion
ATR-FTIR spectra of the samples in the ATR cells were recorded
on PE Spectrum One B instrument. Background was subtracted
using the Opus software. Curve fitting was then performed using
Origin 9.0 and PeakFit v4.12 software. The absorbance spectra of
the samples were recorded by a Hitachi UV9100 spectrophotome-
ter. The range of wavelength is 190–500 nm. The tryptophan (Trp)
fluorescence spectra were recorded by a PE LS55 spectrofluorime-
3.1. Effect of single metal ion on papain activity
The effects of different concentrations of Hg2+ or Cu2+ on papain
activity were investigated and typical low-dose stimulation and
high-dose inhibition (hormesis) was shown in Fig. 1. Hg inhibited
2
+
2
+
papain activity with a relative activity of 6.78% when Hg concen-
◦
−4
ter at 30 C. The emission spectra were recorded in the range of
tration was ≥10 mol/L, but it was observed that stimulation of
−
6
2+
3
00 ∼ 410 nm at 500 nm/min, 10 s after excitation, keeping the exci-
papain activity could occur at 10 mol/L of Hg concentration and
displayed the highest relative activity of 111.10%. There was no sig-
tation constant at 288 nm, with slit widths of 5 nm for excitation
and emission. Tryptophan ethyl ester was used as internal stan-
dard to correct an inner filter effect. The blank spectrum without
enzyme was subtracted from the sample spectra.
−
10
−7
nificant difference in papain activity, exposing to 10
∼ 10 of
2+
2+
Hg . At the same time, the maximum of 58.10% inhibition by Cu
−
4
(41.90% relative activity) was at 10 mol/L, and stimulation with

X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
127
Hg2+ and Cu2+ for the binding sites in papain. One of the most inter-
esting findings was that Cu showed various degrees of inhibition
1
1
50
00
2+
2+
Cu
−7
2+
on papain activity below 10 mol/L Hg , suggesting that papain
was more sensitive to the binary ions. Given all that, the binary
ions were more effective in affecting papain activity than single
2+
Hg
2+
2+
2+
Hg or Cu , though papain activity was entirely inhibited by Hg
−
4
at 10 mol/L. Papain could be adapted to analysis of the binary ions
−
4
with a low detection limit (10 mol/L), a large analytical range and
a short response time. According to the results of the experiment
on papain activity, the Binary union S and I were chosen to inves-
tigate the interactions between the binary ions and papain activity
5
0
&
structure.
3
.3. Influence of binary metal ions on papain secondary structure
0
-11
-9
-7
-5
-3
10
10
Hg2+
10
10
10
mol/L
It was possible that the change in papain activity observed in
the binary ions buffer might be due to secondary structure change
of papain. The changes in papain secondary structure were deter-
mined by ATR-FTIR, UV–vis and fluorescence spectroscopies in the
presence of the Binary union S and Binary union I, respectively.
/
Cu2+concentration
(
)
Fig. 1. Effect of single metal ion on papain activity. ······: Cu2+; —Hg2+
.
−
9
about 131.70% relative activity at 10 mol/L. There was no effect of
Cu below 10
2+
−10
3.3.1. Fluorescence spectroscopy
mol/L on papain activity. In order to see the effect
Fluorescence spectroscopy was used to investigate the pertur-
bation of Trp residues in papain as a result of the binary ions
interaction with papain and the fluorescence emission spectra of
the samples were shown in Fig. 2A. All the samples showed typ-
ical Trp peak excited at about 342 nm and the fluorescence was
mainly due to the presence of five Trp residues in papain [13],
and the maximum emission of the control was at 344 nm (Fig. 2A
of chloride anion, the effect of KCl on papain activity was checked
and there was no change in papain activity (data not shown). Thus
2+
the change in the activity observed was mainly due to the Hg /or
2+
Cu
.
3.2. Effect of binary ions on papain activity
(
a)). Upon interaction of papain with binary union S, the maxi-
mum fluorescence emission appeared a blue shift (6 nm, Fig. 2A
b)) and decrease in intensity compared with the control. This
In order to investigate the interactive effect of the binary ions
on papain activity, the activity of papain at each testing combina-
(
2
+
2+
tion of Hg and Cu was determined, and the binary ions range of
blue-shift could be attributed to the conformational changes in
the vicinity of surface-exposed Trp residues, presumably because
of internalization into a more hydrophobic environment. And the
decreased fluorescence intensities also indicated burying of the
surface-exposed Trp molecules in the interior of the protein. In case
of the Binary union I, the marked decrease in intensity and a red
shift of 3 nm in the emission maximum were found (Fig. 2A (c)).
It might be deduced that the internalized Trp residues in native
state got partially exposed from a hydrophobic to a hydrophilic
environment leading to partial unfolding of the molecule. In the
last case, the Trp residues were buried inside a more polar pro-
tein surrounding, resulting in an inactive enzyme. All these data
clearly suggested that the structural alteration of papain exposed
in binary ions buffers induced the microenvironment change of the
Trp residues. The pattern of the two ions bounded to papain and
located at papain molecular interface/protein surface, this might be
analogous to the bound pattern of cadmium to saru-actinidin [23].
Papaya protease omega (ppꢀ) and saru-actinidin have homologous
overall conformation to papain. ppꢀ has a mercury atom bound to
its active site cysteine, and the NZ atom of Lys64 is 2.88 Å from the
mercury atom of a symmetry-related molecule and this contact
may be important in determining the arrangement of molecules
and hence the space group [18].
− −3
9
1
0
–10 mol/L was based on above results (Fig. 1). The results
were listed in Table 1. The maximum stimulation was 180.80%
relative activity when exposed in Binary union S buffer, showing
that the binary ions enhanced the relative activity by 49.10% com-
paring with the maximum activation of single Hg2+ or Cu . On
the other hand, the maximum inhibition was only 42.8% relative
activity in the presence of Binary union I, which was obviously
2+
2
+
−4
higher than that of single Hg at 10 mol/L on the relative activity
2
+
2+
(
6.78%). Cu was found to suppress the inhibitory effect of Hg
,
and interact antagonistically at most test levels except at combi-
−
9
−5
2+
nations of low concentrations of both 10 ∼ 10 mol/L for Cu
−
6
−5
2+
and 10 ∼ 10 mol/L for Hg , at which they interacted additively.
Results of those experiments ranged from antagonistic to synergis-
2+
tic responses, the overall trend was the addition of Cu reduced
toxicities of Hg on papain. The negative interaction between the
two metal ions was probably the result of the competition between
2
+
Table 1
Effect of the binary metal ions on papain activity at different test combinations.
(Concentration unit: mol/L, papain activity: %)
CCu2+
10
−3
10
−4
10
−5
10
−6
10
−7
10
−8
−9
10
Relativeactivity(%)
CHg2+
−
−
−
−
−
−
−
3
4
5
6
7
8
9
3.3.2. UV–vis spectroscopy
10
10
10
10
10
10
10
40.8
43.4
42.1
76.6
42.3
41.1
40.9
41.1
42.8
55.1
90.6
44.4
44.4
44.2
42.8
42.3
103.5
124.5
47.4
41.1
41.9
112.3
176.5
66.1
42.6
42.6
148.3
170.0
67.0
42.5
41.9
151.2
180.8
82.7
43.0
42.1
146.2
172.9
92.6
Papain has 212 amino acid residues and consists of two domains,
with the active site in the groove between the domains. One
consists of 10–111 and 208–212 residues (l-domain) containing
mainly ␣-helix and another consists of the remaining residues (R-
domain) with a large amount of ␤-sheet. It has 5 Trp residues, 2
of which are located in the ␣-helix segments of the l-domain, and
3 (Trp 7 in the ␤-sheet, Trp 177 and Trp 181 in the coil region) in
the R-domain [6,9]. UV–vis absorption measurement was a simple
47.8
63.6
66.7
62.0
63.9
64.8
95.4
84.5
88.2
78.9
◦
The reactions were performed at 40 C, pH 7.0 for 30 min, 1.0 mg/mL papain Tris–HCl
solution in different concentration combinations of Hg2+ and Cu2+ ions. Every group
test was run three times and the mean values were used as the final test results.

1
28
X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
210 nm
344nm
A
B
338nm
a
b
2
80 nm
c
347nm
c
a
b
320
340
360
380
400
200
250
300
350
400
Wavelength (nm)
Wavelength (nm)
Fig. 2. Fluorescence emissionspectra (A) and UV–vis spectra (B) of the papain samples exposed to Binary union S and I buffer. (a): Control; (b): Binary union S and (c): Binary
union I.
and applicable method to study the binding of small molecule sub-
stances to protein, which was used as a sensitive measure of subtle
changes in protein structure. The UV absorption spectra of papain
and papain-binary ions complexes were investigated and the result
was shown in Fig. 2B. All the samples similarly displayed the dual
absorbance spectra, a strong absorbance with a peak at 210 nm and
a broad band centered around 280 nm, where a weak n → ꢁ* elec-
tronic transition occurred at 210 nm for ␣-helix structure while
a higher frequency ꢁ → ꢁ* electronic transition at 280 nm for Trp
residues [8,14,19]. Clearly, the UV absorption intensity of papain
changed with the variation of the binary ions concentration, but
its shape almost kept in the same. Comparing with the control,
shift of the maximum peak position of papain-binary ions com-
plexes at about 210 nm indicated that the interaction between the
binary ions and papain induced to the change of ␣-helix structure
of papain. Increase of absorption intensity of papain-Binary union
I complex in the region of 280 nm showed that interaction of them
caused Trp residues to migrate towards the protein surface and
increased surface accessibility of Trp. While, the decreased absorp-
tion intensity of papain-Binary union S complex at 280 nm could
be because of the effect of the Binary union S on papain, as a result
of conformational changes in papain structure during the inter-
action of Binary union S with papain, leading to burying insider
a less polar protein surrounding. A similar phenomenon was also
found on Trp residues change in fluorescence spectral investigation
on the secondary structure of papain. Compared with the con-
trol, papain exposed in the Binary union S buffer exhibited the
increases of ␣-helix (from 18.28 to 24.74%) and ␤-sheet contents
(from 22.45 to 28.04%), and decreases of ␤-turn and random coil
contents. Especially, papain showed a decrease in the percentage
of intermolecular ␤-sheet aggregates from 9.68 to 8.89%. However,
the ␣-helix and ␤-sheet contents of papain decreased to 15.66 and
18.27%, while the intermolecular ␤-sheet aggregates contents rose
to 15.05% in the presence of the Binary union I, but there was
only a slight change of ␤-turn and random coil contents. It could
be concluded that the ␣-helix and intermolecular ␤-sheet aggre-
gates contents were mainly contribution to the secondary structure
change of papain. ATR-FTIR spectra revealed that the structure of
papain-binary ions complexes had certain deformation leading to
the structural change of papain, which might be considered as a
significant factor in affecting its catalytic activity. Of the 2 domains
in papain folding structure, l-domain of the native molecule had
large ␣-helix content, while R-domain was mainly ␤-sheet and a
lesser amount of ␣-helix [6,9,21]. Accompanied by the enhance-
ment of papain catalytic activity (Fig. 1), papain presented a modest
increment of ␣-helix content in the presence of the Binary union
S, moreover, slight decrease of intermolecular ␤-sheet aggregates
and random coil contents due to the binding of the Binary union S
to papain at low ions concentrations. Nevertheless, exposed in the
Binary union I buffer, the binding of the Binary union I to papain
decreased ␣-helix and ␣-sheet contents and significantly increased
the intermolecular ␤-sheet aggregates content, leading to inactiva-
tion of papain. The binding of the binary ions to papain was marked
by significant changes of the position in the ATR-FTIR spectra.
With reference to above spectroscopic characterization results,
it was confirmed that the papain activity increased with the
increase of ␣-helix content as well as the decrease of intermolecu-
lar ␤-sheet aggregates content, and vice versa. It was important to
note that efficient catalytic activity of papain required not only cor-
rect active site but also the domain packing properties. Thus these
spectroscopic techniques revealed that the conformation change in
papain upon the interaction of the binary ionS on papain.
(
Fig. 2A), clearly denoting that the interaction between the binary
ions and papain induced a significant structure change which could
be attributed to the papain conformation including ␣-helix struc-
ture. In order to obtain more information on the binding of the
binary ions to papain, ATR-FTIR was employed to study the confor-
mational change of papain.
3.3.3. ATR-FTIR
Deformation in papain secondary structure led to the change of
the protein’s native three-dimensional structure wherein the func-
tion of the protein could also be altered. ATR-FTIR spectroscopy has
been used extensively to study the secondary structure changes
−
1
of protein. The amide I band (1700 ∼ 1600 cm ) was the most
useful for spectroscopic analysis of secondary structure of protein
3.4. Kinetic constants
[
10]. The original ATR-FTIR spectra of the Binary union S and I
−
1
samples in the 1800 ∼ 900 cm region were performed and the
components peaks in amide I region were determined by curve
fitting method (Fig. S1 and Table 2), which the individual compo-
nent locations of the secondary structure were assigned according
to the methods by earlier studies [17,25]. The obtained analytical
result showed that the binary ions buffer had significant influence
The kinetic parameters, Km and Vmax, of the treated papain
were calculated from the Lineweaver–Burk and Michaelis–Menten
models in presence of the binary metal ions (Binary union S and
2
+
−6
−4
Binary union I), single Hg (10
and 10 mol/L) and single
2+
−9
−4
Cu (10 and 10 mol/L) at casein concentrations of 1.0 mg/mL.
Lineweaver–Burk reciprocal plots of the samples were shown in

X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
129
Table 2
Secondary structure assignments and areas in amide I infrared bands of papain samples at Binary union S and I combinations.
Second structure
Binary metal ions concentration (mol/L)
Control
Binary union S
Peak
Binary union I
Peak
Peak
Areas
(%)
Areas
(%)
Areas
(%)
centers
centers
centers
−
1
−1
−1
(
cm
)
(cm
)
(cm
)
Intermolecular
1614
9.68
1614
8.89
1616
15.05
␤
-Sheet
aggregates
␤
␣
-Sheet
-Helix
1627
1655
1642
1669
22.45
18.28
25.22
24.37
1628
1657
1643
1670
1682
28.04
24.74
19.22
19.11
1629
1657
1643
1669
1683
18.27
15.66
26.38
24.65
Random coil
-Turn
␤
1683
◦
◦
The reactions were performed at 40 C, pH 7.0 for 30 min, 0.5 mg/mL papain Tris–HCl solution was equilibrated in Binary union S and Binary union I under 25 C for 10 min,
and then centrifuged at 800 × g for 4 min. The supernatant was used to determine the structure changes of the treated papain. The papain without any metal ions treated
was used as the control. All the samples were determined three times and the data obtained from the triplicate runs were averaged and used as the final result.
ꢀ
Fig S2 and Table S1. For the papain exposed in the Binary union
S buffer, the Vmax value increased to 0.195 mg/min comparing
with that the control (0.121 mg/min), while Km decreased from
could be obtained, and the ratio of k0 to k0 was 0.2954, which
suggested that there was a binding of the binary ions (Y) with
both the native enzyme (E) and the enzyme-substrate complex
(EY), and amount of EYS was more than that of EY. Additionally,
4
.733 to 3.663 mg/mL (Fig S2 A). On the contrary, the Vmax value
of the papain exposed in the Binary union I buffer reduced to
.067 mg/min, while Km climbed to 5.163 mg/mL (Fig S2 B). Simi-
2
a plot of 1/A versus [S] gives a nearly liner (R = 0.9131), and A
0
value increased with increasing substrate concentration [S], imply-
ing the Binary union S was mainly competitive activator for papain
although noncompetitive stimulation also occurred. In our pre-
2+
lar experiments were carried out in the presence of single Hg and
2+
Cu , and the values of Km were determined, listing in Table S1. The
values of Km indicated the high affinity for casein in the presence of
the Binary union S, and low affinity in the Binary union I. Higher Km
−
6
2+
vious work, 10 mol/L Hg was a noncompetitive activator for
2+
papain, the binding interactions of Hg with papain mainly located
outside the active center . Based on the kinetic parameter Km
results (Table S1), the order of the affinity for the substrate was
2
+
−6
−4
2+
values for Hg at 10 and 10 mol/L than for Cu corresponding
−
9
−4
at 10 and 10 mol/L indicated the papain had a lower affinity for
the substrate, displaying that the affinity of the papain bound with
−
9
2+
−6
2+
−4
Binary union S > 10 mol/L Cu > 10 mol/L Hg > 10 mol/L
Hg for the substrate was lower than one bound with Cu2+. The
papain exposed in the Binary union S buffer had the smallest Km
value, implying that it had the highest affinity for the substrate. The
Km value of the papain exposed in the Binary union I was similar to
2+
7
6
Control
1.2
2
+
−4
2+
the one with Cu at 10 mol/L, but much higher than with Hg
at 10 mol/L, indicating that addition of Cu blocked the inhibi-
tion of Hg on papain. The result showed that the Binary union
I was able to weaken the affinity for the substrate by decreasing
−
4
2+
5
4
3
2
1
0
.8
.4
2
+
0
␣
-helix content and enhancing intermolecular ␤-sheet aggregates
content.
0
.0
.2
7
.5. Kinetics properties of papain in the presence of Hg2+
Binary union S
Binary union I
3
1
6
5
The time course of hydrolysis of casein in different binary ions
0.8
concentrations was shown in Fig. 3. For the substrate hydrolysis
4
2
+
2+
in the presence of Hg + Cu , the rate increased with increasing
casein concentration, while the slope of the asymptote increased
with increasing casein concentration. The reaction progress curves
of the control were linear over a lengthy period of time. The results
analyzed by Zhou and Tsou’s method [24] suggested that the Binary
union S had a stimulating effect on papain activity, but the Binary
union I had an inhibition effect. According to Eq. (1), the exponential
linearized expressions had been achieved using least square fitting
method by all data in Fig. 3, and the kinetic parameters of the model
were shown in Table 3. The results showed that the calculated the
3
0.4
2
1
0
.0
.2
1
0.8
.4
0.0
7
6
5
4
3
2
0
2
correlation coefficients R were greater than 0.974, indicating that
the kinetic model could well describe casein hydrolysis during the
1
binary ions binding to papain. According to Eq. (3), the plot of 1/[Pe]
10
20
30
40
50
60
ꢀ
against 1/[S] gave a straight lines. The values of k0 and k0 were
Time (min)
calculated, and listed in Table 3.
ꢀ
The v value of native papainwas lesser than the v value of
Fig.3. Time course of casein hydrolysis reaction at different substrate concentra-
tions in the presence of Binary union S and I buffer. (1) 0.2 mg/mL casein; (2)
.4 mg/mL casein; (3) 0.6 mg/mL casein; (4) 0.8 mg/mL casein; (5) 1.0 mg/mL casein;
(6) 1.4 mg/mL casein; (7) 2.0 mg/mL casein. ······: Prediction; ꢀ: experiment.
papain exposed in the Binary union S buffer, and this indicated
that the Binary union S could stimulate papain catalytic activ-
0
ꢀ
ity. Furthermore, the values of dissociation constant k0 and k0

1
30
X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
Table 3
Kinetic parameters and dissociation constant of papain samples at the two Binary union S and I combinations.
ꢀ
R2
ꢀ
No.
Casein concentration
mg/mL)
A
ꢂ
V
[Pe]
(mg/mL)
k0
k0
(
(mg/mL)
(mg/min)
(mg/min)
Control
0.2
–
–
–
–
–
–
–
0.0028
0.0028
0.1984
0.3908
0.5809
0.7082
0.8687
1.0824
1.3465
0.1135
0.2195
0.3066
0.4394
0.6078
0.7070
0.8423
0.1987
0.3936
0.5883
0.7243
0.8927
1.1022
1.3871
0.987
0.990
0.992
0.992
0.996
0.996
0.999
0.987
0.989
0.992
0.974
0.985
0.996
0.993
0.986
0.994
0.994
0.996
0.997
0.998
0.998
0.4
0.6
0.8
1.0
1.4
2.0
0.00549
0.00815
0.00889
0.00944
0.01111
0.01211
0.00176
0.00594
0.00756
0.01075
0.02118
0.01913
0.02677
0.00469
0.01097
0.0131
0.00549
0.00815
0.00889
0.00944
0.01111
0.01211
0.00664
0.00683
0.00978
0.03342
0.05733
0.05619
0.07482
0.00106
0.00023
0.00026
0.00203
0.00313
0.00667
0.01017
–
–
Binary union I
Binary union S
0.2
0.00863
0.05267
0.07716
0.00850
0.00673
0.00858
0.00752
0.01222
0.01499
0.01647
0.01852
0.01834
0.07880
0.11128
0.4
0.6
0.8
1.0
1.4
2.0
0.114
0.688
5.493
2.329
0.2
0.4
0.6
0.8
1.0
1.4
2.0
0.0113
0.01181
0.01347
0.01604
The reactions were performed at 40 C, pH 7.0 for 30 min, 1.0 mg/mL papain Tris–HCl solution in different concentration combinations of Hg2+ and Cu2+ ions. Every group
◦
test was run three times and the mean values were used as the final test results.
Cu2+ ≈ Binary union I > 10 mol/L Hg2+. In the Binary union S
buffer, papain had two sites of combination for the activator, one
in the active pocket and the other one on the surface of enzyme.
−4
ate from the metal binding sites of papain and therefore much less
effective than Cu2+ in the inhibiting effect.
2
+
Since Cu had higher affinity to papain, it may insert into the
4. Conclusion
2
+
active pocket, while Hg bond with the binding site of the surface
to some extent. The binding of Binary union S to papain induced
papain conformation to change especially due to the decrease
of intermolecular ␤-sheet aggregates content (Table 2), so that
the hydrophobic pocket became bigger. It could be hypothesized
that the combination of the substrate with the enzyme molecule
induced a broadened hydrophobic pocket in the enzyme-substrate
complex, and the combination of the substrate with papain
molecule would be easier to engender, leading the increase of
papain activity.
The study demonstrated that there were significant impacts of
the binary ions (Hg & Cu ) on papain. The strongest inhibitive
2+
2+
2+
2+
effect was observed when Hg and Cu concentration was up
−4
to 10 mol/L, whereas maximum activation concentration was
−8 −6
2+
2+
1
0
mol/L Cu + 10 mol/L Hg . So, the low detection limit for
−4
2+
2+
papain was 10 mol/L concentration of Hg + Cu . The results
showed that the performance of papain was suitable for the
quantitative determination of the binary ions in environmental
analysis.
ꢀ
The v value was higher than the v value in the Binary union I
The fundamental correlations between the structure of papain
and its catalytic activity were clarified. The three-dimensional
structure of the papain exposed in the binary ions was determined
by ATR-FTIR, UV–vis and fluorescence spectroscopies. The papain
exposed in the Binary union S buffer had increase in ␣-helix content
and decrease in intermolecular ␤-sheet aggregates content, but the
papain exposed in the Binary union I buffer had increase in inter-
molecular ␤-sheet aggregates but decrease in ␣-helix content. The
papain activity increased with the increase of ␣-helix content and
decrease of intermolecular ␤-sheet aggregates content, and vice
versa.
buffer, revealing that the Binary union I was an inhibitor for papain.
ꢀ
The ratio of k to k was 1.8028, showing that binding of the Binary
0
0
union I with papain also had two existing form, EY and EYS, but the
amount of EY was more than that of EYS. The apparent rate con-
stant A was independent from [S] (Table 3), namely the inhibition
had nothing to do with the substrate concentration, implying that
the Binary union I was noncompetitive inhibitor for papain and
could bind with papain at the active sites both in the active pocket
and on the protein surface. Results from those experiments ranged
from inhibitory to synergistic responses (Tables 1 and 3), though the
2
+
2+
overall trend was for Cu to reduce toxicities of Hg on papain.
More experimentation was necessary to better understand the
mechanisms underlying the interactions between heavy metals
2+
2+
The negative interaction between Cu and Hg was probably the
result of the competition between Cu2+ and Hg2+ at the binding
sites of papain.
2+
2+
such as Hg & Cu , and it was evident that the interactions of the
binary ions affected papain activity considerably. There were dif-
ference interaction mechanisms between the binary ions and single
As described in our previous work (data not shown), 10 4 mol/L
−
Hg2+ as competitive inhibitor was found to mainly bind at the
2+
Hg . The Binary union S as a competitive stimulator was better able
2+
active sites in papain active pocket. Since Hg was a typical soft
acid, and papain was one of cysteine proteinases should contain
soft bases, i.e., cysteine residues, Hg2+ had a strong bind with the
residues resulting in almost full deactivation of papain. The inhibi-
tion of Hg could be recovered in the presence of Cu (Table S1).
Cu with higher affinity for papain could recover papain activity
by competitively binding to the active sites, which leading to the
decrease of the inactive sites for Hg . It was speculated that the
active sites should favor small metal ions. Since Hg had a much
larger radius than Cu , it was reasonable that Hg might devi-
2+
−6
2+
to enhance papain activity than single Hg at 10 mol/L or Cu
−9
2+
2+
−4
at 10 mol/L. For single Hg , the Hg at 10 mol/L was a com-
petitive inhibitor and papain activity was almost lost completely,
suggesting that the SH group situated at the active site in active
packet of papain was essential to maintain its catalytic activity and
2
+
2+
2+
2+
the reaction of Hg with SH group led the enzyme activity to lose
entirely. However, the inhibition of papain by the binary ions in
2+
2+
the Binary union I buffer belonged to be noncompetitive type. Cu
2
+
bonded preferentially with papain into the active packet by remov-
2
+
2+
2+
2+
ing Hg leading to the mostly binding of Hg with the cysteine

X.-Y. Liu et al. / Biochemical Engineering Journal 97 (2015) 125–131
131
residues (SH group) outside of papain active packet. Regardless
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