Study the effect of trypsin enzyme activity on the screening of applying frontal affinity chromatography

Article abstract of DOI:10.1016/j.ijbiomac.2019.07.218

Frontal affinity chromatography (FAC) combined with enzyme has been widely used for drug screening. In this paper, the effect of target enzyme activity on screening of bioactive compounds was studied through applying FAC. Trypsin with different degree of inactivation were prepared as target enzyme by thermal denaturation. Their primary structure was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and use Fourier transform infrared (FTIR) and ultraviolet-visible (UV–vis) spectroscopy to detect group structure. Ultimately, it was found that the main structure of enzyme with decreased activity remained unchanged. The oxymatrine and matrine which can interact with trypsin were selected to study their binding to trypsin with different activities in FAC. The results showed that oxymatrine and matrine had a significant difference in the breakthrough volume among seven kinds of columns prepared by trypsins with different activities, at the different concentration. It indicated that trypsins with different activities in FAC could combine with oxymatrine and matrine. The binding constant (K_d) variation between oxymatrine, matrine and trypsin with different activities are 5.520 ± 0.038 and 3.577 ± 0.071, within error range, which indicated that the activity of target enzyme with primary unchanged structure has no effect on screening of bioactive components by FAC.

Full text of DOI:10.1016/j.ijbiomac.2019.07.218

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Study the effect of trypsin enzyme activity on the screening of
applying frontal affinity chromatography
JunQing Qian, ChangYan Zhao, Jun Tong, ShengLan Jiang,
Zheng Zhang, ShiYong Lu, Hui Guo
PII:
S0141-8130(19)34777-4
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.07.218
BIOMAC 12970
Reference:
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
25 June 2019
30 July 2019
31 July 2019
Please cite this article as: J. Qian, C. Zhao, J. Tong, et al., Study the effect of trypsin
enzyme activity on the screening of applying frontal affinity chromatography,
International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/
j.ijbiomac.2019.07.218
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Study the effect of trypsin enzyme activity on the screening of applying frontal
affinity chromatography
JunQing Qian*, ChangYan Zhao, Jun Tong, ShengLan Jiang, Zheng Zhang, ShiYong
Lu, and Hui Guo
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou
310014, China
*To whom correspondence should be addressed: JunQing Qian, College of
Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014,
China;
E-mail: junqingqianzjut@163.com

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Abstract
Frontal affinity chromatography (FAC) combined with enzyme has been widely used
for drug screening. In this paper, the effect of target enzyme activity on screening of
bioactive compounds was studied through applying FAC. Trypsin with different
degree of inactivation were prepared as target enzyme by thermal denaturation. Their
primary structure was identified by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and use fourier transform infrared (FTIR) and
ultraviolet-visible (UV-vis) spectroscopy to detect group structure. Ultimately, it was
found that the main structure of enzyme with decreased activity remained unchanged.
The oxymatrine and matrine which can interact with trypsin were selected to study
their binding to trypsin with different activities in FAC. The results showed that
oxymatrine and matrine had a significant difference in the breakthrough volume
among seven kinds of columns prepared by trypsins with different activities, at the
different concentration. It indicated that trypsins with different activities in FAC could
combine with oxymatrine and matrine. The binding constant (Kd) variation between
oxymatrine, matrine and trypsin with different activities are 5.520±0.038 and
3.577±0.071, within error range, which indicated that the activity of target enzyme
with primary unchanged structure has no effect on screening of bioactive components
by FAC.
Keywords: trypsin enzyme activity, frontal affinity chromatography, screening.

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1. Introduction
Enzymes, that play an important role in pathological research, have been
extensively used to screen bioactive compounds from complex natural medicines as
molecular targets [1]. Current methods of screening with target enzymes are mainly
about investigating the inhibitory effect of screened target on enzyme activity [2], thus
it is reasonably restrict to target enzyme activity. Most of the target enzymes used in
the screen process are extracted from patients’ diseased tissue, so it is extremely
precious. While target enzymes cloned by genetic engineering tend to be less active [3,
4], it is difficult for screening according to existing technology. Consequently, it is of
great significance to study a drug screening method that does not require molecular
target with high enzyme activity.
Frontal affinity chromatography (FAC) is a method for quantitative analysis of
biomolecular interactions [5-7]. It was established by Kasai and Ishii in 1975 [8].
FAC has been widely used in drug screening such as kinase screening, target for drug
molecules, and leading compounds optimization [9-11]. FAC achieves the screening
of drugs by specific affinity adsorption between bioactive compounds and targets on
the surface of the stationary phase [12]. FAC-based methods have the advantage of
being easy-to-be reproducible, that is, columns can be regenerated by eluting of the
original application buffer, and are able to identify not only strong (nM range) binding
molecules but also weak binding molecules (mM range) [13, 14], which can offer
high precision and accuracy in the measurement of binding constants and realize high
throughput screening [15]. With the development of modern molecular biology,

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biomacromolecules with important physiological function such as enzymes, receptors
and DNA could be used as ligand of affinity chromatography [15-18], which can be
used to simultaneously characterize single compounds in complex chemical mixtures
[19, 20] and to isolate and identify active components from complex biological
matrices [19-21] due to its simplicity, flexibility, selectivity, and easy-to-be use [14].
For example, a library of 356 β-galactopyranoside compounds were divided in a
group of 25-40 compounds which were screened using an immobilized
β-galactosidase enzyme on a LC stationary phase column [10]. It can be adapted to
potentially isolate new, previously unknown and therapeutic drugs [22]. FAC offers
new entry points including dual binding site assay, receptor subtype characterization
and multi-receptor binding experiments. Moreover, FAC method can be easily
extended to receptors bearing mutations in key amino acids for ligand recognition
[15].
The technique of target enzyme immobilization combined with FAC has already
been applied to drug screening [23, 24]. Compared with free enzyme, immobilization
can improve stability, recovery and its facile separation from the product [25, 26]. The
use of a polymer material with good bioaffinity to immobilize the target enzyme is the
basis for effective screening. Pinheiro used the chitosan carrier activated with divinyl
sulfone to immobilize the enzyme and found that this method can improve the
stability of the enzyme [27]. The requirement for enzyme immobilization in FAC is
quite different from that of the enzyme as a catalyst. It is required that the enzyme
should be stably immobilized in the column without catalytic reaction. The steric

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hindrance is mainly responsible for the binding of the target enzyme to the screening
compound. Therefore, the interaction between target enzymes and bioactive
compounds in FAC is mainly related to the structure of enzymes, especially primary
structure [28]. However, there are few researches about the effect of target enzyme
activity on the screening for bioactive compounds. And the target enzyme activity is
closely related to its molecular spatial structure.
In order to explore whether the results of screening active compounds using
enzyme as the target is connected with the activity of target enzymes, we have chosen
trypsin as target enzyme and applied FAC to carry out screening test in this paper.
There are many research reports on the value of trypsin as a target for drug screening
[29-31], and immobilized trypsin is also an important tool for protein research
[32].We have previously conducted some studies about immobilized trypsin
commbined with FAC to screen matrine and oxymatrine, which was published in the
literature [33, 34]. The results showed that trypsin immobilized with
glycerol-modified tetraethoxysilane was reliable for FAC screening. The binding of
matrine and oxymatrine to trypsin was specific. Therefore, the correlation between
target enzyme activity and screening effect has a good foundation. Trypsin with the
same primary structure but different enzyme activities was prepared through thermal
denaturation, which was identified by SDS-PAGE, FTIR and UV-vis spectroscopy,
and their structure are consistent with purchased original enzyme. Matrine and
oxymatrine were selected as the test analyte [33]. Trypsin frontal affinity
chromatography with different enzyme activities was prepared to detect the binding

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constants (Kd) between the test analytes and trypsin respectively.
2. Materials and methods
2.1 Materials
(3-Aminopropyl)triethoxysilane (APTES), tetramethylammonium chloride
(TMAC)， poly ethylene glycol 600 (PEG600) were purchased from Acros Organics.
Trypsin was a commercial enzyme available from Amresco and it is of biochemical
reagent grade. Trypsin (EC 3.4.21.4) is single-point pure by SDS-PAGE detection.
Bovine serum albumin, which is of electrophoresis single point pure, was purchased
from Lanji Technology Development Co., Ltd. in Shanghai. Amberlyst 15 (dry) was
purchased from Aldrich Chemical Co. Matrine and oxymatrine (HPLC grade, 98.5%)
were purchased from Tontian Bio Co., Ltd. in Shanghai. Ethyl N-benzoyl-L-argininate
hydrochloride (BAEE) was purchased from Sinopharm Chemical Reagent Co. Ltd.
Other chemicals were analytical grade reagents and double distilled water was used
throughout.
FAC system was equipped with LSP-1B Longer Pump and TS2-60 Dual-syringe
Pump (Longer, China), and N2000 chromatographic workstation (Zhejiang
University). Fused-silica capillary (700mm i.d.) and a capillary connectors were
purchased from Innosep (China). DYCE-24DN vertical electrophoresis apparatus and
DYY-10C electrophoresis apparatus were made by Beijing Liuyi Bio Co. Ltd. Tensor
27 FTIR spectrometer (Bruker, Germany) and Evolution 300 UV-Vis
spectrophotometer (Thermo Scientific, USA) were used. HitachiS-4700(II) Field
Emission Scanning Electron Microscope (Panasonic, Japan) and AVANCE III

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500MHz Nuclear Magnetic Resonance Spectrometer (Bruker, Germany) were used.
2.2 Glycerol modification of tetraethoxysilane
Tetraethoxysilane (TEOS) can be used as a material to immobilize trypsin in
preparing frontal affinity chromatography column. TEOS was modified by glycerol,
namely, oxethyl of TEOS was replaced by hydroxyl group of glycerol, so as to
improve its affinity to target enzymes and hydrophilicity, which was beneficial for
preparing chromatography column [34].
According to the method described in the previous study [35], 3.0 g of Amberlyst
(dry) strongly acidic cation exchange resin as catalyst and 50.0 mL TEOS were added
into a 250 mL three-necked flask, and the mixture was stirred at 180 r/min under 50
°C water bath. Then, glycerol was added in the form of drop (TEOS: glycerin=1: 0.25,
n/n), and the reaction was carried out for 72h. After the completion of reaction, the
catalyst was filtered off. Subsequently, the filtrate was distilled away under reduced
pressure at 25 °C for 8 h (vacuum degree less than 30 hPa), and transparent solid gel
was obtained.
The above glycerol-modified tetraethoxysilane is polymerized through silane
coupling agent APTES to form a gel that can immobilize Candida rugose lipase. The
immobilized enzyme has higher catalytic activity than free enzyme in an organic
solvent, indicating modified TEOS has good biological affinity [35].
The trypsin immobilized with glycerol-modified TEOS has an immobilization
yield of about 67%. Compared with the free enzyme, the optimum temperature
increased to 70 ° C and the suitable pH tends to be neutral. The storage stability is

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significantly improved at room temperature The enzyme activity is basically not
changed in 30 days. The properties of the above modified TEOS-immobilized trypsin
has been published in the literature [36]. Dos Santos found that pH in the
immobilization has an impact on immobilization yield, which is very consistent with
the result conducted by our laboratory [37].
The glycerol-modified tetraethoxysilane was characterized by infrared
spectroscopy, 13C-NMR and 1H-NMR.
2.3 Activity detection of trypsin
Activity of trypsin was detected by the method: BAEE units/mg [38]. BAEE was
used as substrate，and its concentration was 0.34 mg/mmL in Tris-HCl buffer
solution (0.05 mol/L, pH8.0 ). After adding the enzyme, BAEE was hydrolyzed into
N-benzoyl-L-arginine(BA) at 25℃, which was analyzed by UV absorbance at 253 nm.
The activity of trypsin was calculated as follows:
(which m was enzyme dosage) (1)
2.4 Primary structure identification of heat inactivated trypsin
2.4.1 SDS-PAGE
0.004 g of trypsin dissolved in 120 μL of redistilled water, which was divided
into 8 groups and numbered (from 1 to 8). Group 2 to 4 were heated at 50oC for
20min, 30min, 40min. Group 5 was heated at 60oC for 20min. Group 6 to 8 were
heated at 70oC for 20min, 30min, 40min, respectively. And Group 1 was untreated.
Group 0 is an electrophoretic single-point purity of bovine serum albumin, which was

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used as a reference. Then, trypsin solution (15 μL) and loading buffer (3 μL) were
added into each hole of the gel. The power was turned on at the voltage from 80 V to
100 V until bromophenol blue approached to the bottom of the gel. After
electrophoresis, the gel was dyed in Coomassie Brillant Blue G-250 for 2 h. Then, the
gel was discolored in EtOH/acetic acid/H2O (4:1:5, v/v/v) for 4-8 h.
2.4.2 FTIR
0.4 g of trypsin dissolved in 12 mL of redistilled water. Samples were divided
into 8 groups, and the thermal denaturation of trypsin was the same as 2.4.1. The
IR-spectra were recorded in the transmission mode with a Tensor 27 Fourier
Transform Infrared Spectroscopy (Bruker, Germany). The measurements were
performed in a transmission mode using a demountable thin layer IR-cell, with KBr
windows in the frequency region 500-4000 cm-1. Each spectrum was recorded at a
resolution of 4cm-1, 200 scans were averaged before Fourier transformation, taking
the KBr spectrum as background. After background correction, all spectra were
baseline corrected, and then trypsins spectra were obtained.
2.4.3 UV-vis absorption measurement
Based on 2.4.2, after thermal denaturation, the absorbance measurements were
performed through Evolution 300 UV-Vis spectrophotometer (Thermo Scientific,
USA). The UV-vis absorption spectra were recorded at a scanning speed of
400nm/min on spectrophotometer with a slit of 2 nm. The wavelength was selected
between 200-600 nm. The UV-vis spectra were collected with collecting for
background (quartz cell) spectrum, and the UV-vis absorption spectra were collected

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every 1 nm.
2.5 Preparation of FAC column
According to the method of chromatographic column preparation from the
screening platform established by our laboratory[27], the fused-silica capillary
(15cm,50μL) was first washed with 5 column volumes 1M NaOH solution, 1M HCl
solution, H2O and absolute ethyl alcohol (EtOH), respectively at room temperature.
And then the capillary was sonicated in 10 wt% APTES in EtOH solution for 40 min.
Finally the capillary was washed with absolute EtOH and dried at 40 oC.
The immobilized trypsin columns was prepared as follows. First, 45 mg of
trypsin was added into 128.2 μL of redistilled water at room temperature, followed by
120 μL of （200mg/mL）PEG 600, 50 μL of 0.2M phosphate buffer (pH 7.0), and 1.8
μL of（10%，v/v） APTES to form 300 µL aqueous solution, which was stored at 4oC
until the enzyme dissolved in this solution (solution A). 0.1 g glycerated silane, 0.15
mL double distilled water and 2 μL 1M H3PO4 were added together, and then
sonicated to produce a homogeneous solution (solution B). Mixing solution A with B
in equal volumes. The enzyme concentration is 75 μg/μL (pH 7.0, 7μM phosphoric
buffer solution). Then the mixture was injected into the pretreated capillary via
syringe pump within 3 min, solidifying at room temperature. Then the column was
stored in PBS(pH 7.0) at 4oC for 7 days until use.
The cross section of the column was detected by scanning electron microscopy to
characterize the immobilization of trypsin in the column.
Monolithic column was used to prepare FAC column, which makes the

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performance of the modified sol-gel immobilized enzyme better. The pH of the gel
was established by the performance of the column, and the enzyme activity of the
immobilized enzyme decreased by less than 5% at 4 ℃ after 30 days. PEG 600 was
used as the porogen. The aging temperature at 40 °C and the aging time for 7 days
make the column with good permeability. Meanwhile, the loading yield of column is
22.2%, which gives the best preparation conditions for the column. The particle size
of the gel particles aged for 14 days is about 1 μm through SEM. The gel is
continuous when the aging time is short. 50 mM tetramethylammonium chloride was
added in the eluent to eliminate non-specific affinity, which is similar to the results
reported by Dos Santos [37]. The relative standard deviation (RSD) between runs was
in the range of 1%. Furthermore, the RSD was 5% after consecutive analysis for 9
days [34].
2.6 FAC Assay
Oxymatrine/matrine was dispersed in 0.05M of phosphate buffer (pH 7.0,
containing 0.1M TMAC) at concentrations of 10μM、2μM、0.4μM、0.08μM. The
column was washed with 1mL of 0.05M phosphate buffer at pH 7.0 to remove any
residual glycerol, PEG, or loosely bound protein. The system was operated by double
syringe pumps which contained test sample and elution buffer, respectively. The
elution buffer flowing through the immobilized affinity column was detected by
UV-Vis spectrophotometer (Shimadzu SPD-20A, equipped with a semi micro flow
through cell). The two syringe pumps contain test sample and elution buffer
respectively. All solutions were infused at a flow rate of 4 µL/min. The elution curve

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of test sample was determined by UV-vis spectrophotometer at the maximum
detectable absorption wavelength. The maximum absorption of matrine and
oxymatrine were 227nm and 228nm, respectively. The column was re-equilibrated
with elution buffer by switching the loading valve. Then the samples were tested in
frontal mode at various concentrations. Breakthrough volumes were measured as
midpoints in the extracted chromatograms. After the test was completed, the column
was regenerated with phosphate buffer (0.05M, pH 7.0) and stored in phosphate
buffer solution at 4oC. Breakthrough volumes were measured as midpoints in the
extracted chromatograms. All data were processed with Microsoft Excel software and
the frontal chromatograms are presented as N2000 program files.
Trypsins with different activities can be used to prepare different columns. The
binding constant Kd between target enzyme and sample could be calculated through
Eq. (2), according to elution curve and breakthrough volume.
The dissociation constant (Kd) was determined by using the FAC theory of
Kasai[8]. The relationships of the Kd, the column capacity (Bt), an analyte (A) and an
immobilized ligand (B), the initial concentration of A ([A]0), the elution volume of A
(V) are given by equation (2)
(2)
For the determination of Bt and Kd, equation (2) is modified into equation (3), by
using various [A]0, Bt and Kd can be easily obtained from this double reciprocal plot
(1/[A]0 (V -V0) vs. 1/[A]0)

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(3)
In order to eliminate non-specific interaction with the adsorbent, we applied
phenol as blank solvent which showed no affinity to trypsin. The breakthrough
volume of 0.0188mL was regarded as V0 throughout the series of experiments.
3. Results and discussion
3.1 Characterization of glycerol modified tetraethoxysilane
The chemical structure of the glycerol-modified tetraethoxysilane was
investigated by 13C-NMR and 1H-NMR, and the results are shown in Figure 1.
According to Figure 1 (A), the spectra exhibited the typical peaks of ethoxy
including the signal of protons at 1.2 ppm and 3.85 ppm, glycerol absorption peak is
at 3.5-3.6 ppm, and a new absorption peak appears at 3.6-3.8 ppm. New peak is a
characteristic peak of a hydrogen atom on the carbon attached to the oxygen,
indicating that the glycerol hydroxyl group replaces the ethoxy group on the TEOS.
According to Figure 1 (B), the spectra exhibited the typical peaks of ethoxy
including the signal of carbon at 18.4 ppm and 59 ppm, glycerol absorption peak is at
63 ppm and 74 ppm. And a new absorption peak appears at 60 ppm and 73 ppm,
which can confirm the reaction of glycerol-modified tetraethoxysilane.
The functional group present on glycerol modified tetraethoxysilane were
analyzed by FTIR spectroscopy as shown in Figure 2.
It can be found that the infrared spectrum of the glycerol-modified TEOS has
strong absorption at 3600-3300 cm-1 in Figure 2 (B), indicating that the product has

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more hydroxyl groups. The infrared spectrum of TEOS was weakly absorbed at
3600-3300 cm-1 in Figure 2 (A), indicating that the ethoxy group of TEOS was
replaced by the hydroxyl group of glycerol, confirming the modification reaction.
3.2 Trypsin activity after heating at different temperatures
BAEE (0.34 mg) and CaCl2 (2.22 mg) were dissolved in 50 mM Tris-HCl buffer
(pH 8.0). 0.5 mg.mL-1 of trypsin was heated for 20min, 30min, 40min at 50oC, 60oC,
70oC, respectively. Then 0.3 mL of above enzyme solutions were mixed with 2.7 mL
of BAEE solution which was incubated at 25oC respectively, measured the UV
absorbance at 253 nm every 30s with a total of 12 times (the UV absorbance of the
mixture of 0.3 mL 0.001M HCl and 2.7 mL BAEE was set to zero). The activity of
trypsin was obtained by using Eq. (1). Results were shown in Table 1, and the
original activity of trypsin is 19555.6±1.2U/g.
As is shown in Table 1, the activity of trypsin decreased significantly (about 35%
of the original activity) after heating at 50 ℃ for 40min. The activity decreased
substantially when it was heated to more than 60 ℃, and the minimum activity was
only around 1% of the original activity. Thus, a series of trypsin with different
activities were obtained through heating, for further detection of primary structure.
3.3 Identification of trypsin primary structure
3.3.1 SDS-PAGE
Polyacrylamide gel electrophoresis (PAGE) is an efficient method for qualitative
analysis of enzymes and it also can be used to determine their molecular weight. Its
molecular weight would change when the primary structure of zymoprotein changed

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[39, 40]. The molecular mass of trypsin is 24 kDa. As shown in Figure 3,
electrophoretic bands of inactivated and original enzymes were at the same level.
Since the electrophoretic mobility of protein-SDS complex is only related to
molecular weight, it would not be affected by the original charge and molecular shape
of protein. The activity of trypsin changed significantly after heating, while the
electrophoretic band shows that there is no obvious change in molecular weight,
which indicated that the primary structure of trypsin was unchanged.
3.3.2 FTIR
FT-IR spectra of different activity of trypsin were illustrated in Figure 4. The
carboxyl group was presented at 3456cm-1. The fraction of peptide bonds in α-helical,
β-sheet, and aperiodic conformations can be accurately estimated by analysis of the
amide I band (1600-1700cm-1) in the mid-IR region [41]. The amide I band had a
characteristic shape for each peptide, which had the maximum of absorption at
1642cm-1. Red shift and blue shift were not found, indicating groups in trypsin were
unchanged. So, the secondary structure had not changed. The secondary structure of a
polypeptide chain is mainly determined by polypeptide composed of backbone
peptides folding into a regular and cyclical space-conformation, such as α-helical,
β-sheet [42]. Thus, with unchanged groups and secondary structure, we could infer
that the primary structure of trypsin was unchanged.
3.3.3 UV-vis
UV-vis absorption spectra was performed to test whether there exists the change
of groups in trypsin during thermal denaturation. From Figure 5 (1), it is known that

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trypsin shows a great UV absorption at 230nm and 280nm. The absorption at 230nm
was mainly caused by n-π* transition of C=O in peptide bond, and the absorption at
280nm was mainly caused by n-π* transition of heteroaromatic ring in tryptophan and
tyrosine residues. While after thermal denaturation, the UV absorption pear were still
at 230nm and 280nm, that is, red shift and blue shift were inexistent. So we could
infer that trypsin structure was unchanged.
3.4 Scanning electron microscopy of trypsin frontal affinity chromatography
column
The surface morphology of the trypsin front affinity chromatography column
was observed using SEM in Figure 6.
It can be seen from Figure 6 (2) that the structure of the modified silica gel has a
basic network shape after aging for 7 days, which is similar to the report in the
literature [43]. It can be found that as the aging time increased, the silica gel to
immobilize trypsin gradually forms a network. The morphology changes in the silica
gel in the frontal affinity chromatography column are basically similar with the result
reported in the literature [38]. However, the aging time of the column prepared in the
literature is longer [38], and the silica gel inside the column forms a network with
obvious pores after aging for 14 days. It shows that the silica gel structure inside the
column has no obvious pores in Figure 6 (2). Compared with the column with longer
aging time, the sample will go through the column slowly that is beneficial to improve
the binding between target enzyme and the active compound.
3.5 Analysis of interaction between oxymatrine, matrine and trypsin with

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different activity
Based on the theory of FAC, the values of binding constant (Kd) and the column
capacity (Bt) can be determined by breakthrough volume. If the breakthrough volume
V of each sample are the same, and at the same time, they are less than V0, it indicates
that this compound has no affinity with the enzyme. Conversely, if the breakthrough
volume V of each sample are different, and greater than V0, it is indicated that the
compound has an affinity with the enzyme. Meanwhile, the smaller the Kd is, the
greater the binding between enzyme and compounds is. Therefore, the target
compounds with strong affinity to enzymes will be screened [6].
Columns with trypsins activities 19555.56U/g, 13688.90U/g, 9022.22U/g,
6866.67U/g, 2488.89U/g, 444.44U/g, 222.22U/g were prepared and aged, according
to the preparation of 2.3. Preparing standard solutions of oxymatrine and matrine,
which concentrations were 10μmol/L、2μmol/L、0.4μmol/L、0.08μmol/L, respectively.
The maximum absorption of oxymatrine and matrine were 228nm and 227nm
respectively by full wave scanning of UV spectrophotometer (Figure 7).
The values of Bt and Kd of oxymatrine and matrine (Table 2, where x=[A]0 (μM)
and y=1/(V-V0) (mL-1)) in different affinity columns were calculated by Eq. (3), and it
could be compared for the binding between samples and columns with different
activities of trypsin clearly by Bt and Kd. As the concentration of ligand was increased,
the column would saturate more rapidly at a given flow rate. Breakthrough curves of
oxymatrine and matrine are shown in Figure 8 and Figure 9. Small breakthrough
volumes were obtained at high concentration of samples, in affinity columns with

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different activities of trypsin.
The Kd values between oxymatrine and original target enzyme or inactivated
enzymes was 5.600±0.000 and 5.520±0.071, respectively. Similarly, the Kd values
between matrine and original target enzyme or inactivated enzymes was 3.890±0.000
and 3.577±0.038, respectively. Comparing the Kd values of two samples which were
measured in inactivated target enzymes-based columns with original enzyme-based
column, it was found that the value of Kd decreased slightly. Results indicated that the
combination of oxymatrine and matrine with trypsin is slightly influenced by the
changes of enzyme activity.
Although the activity of target enzyme changed, the primary structure remained
unchanged, and it might be inferred that the binding of sample and target enzyme was
a single-site binding, which was mainly related to the primary structure of enzyme.
Meanwhile, the combination of the two is mainly caused by the interaction between
different groups, which is not closely related to the spatial structure of the enzyme.
From the Table 2，the Kd value of matrine was lower than oxymatrine indicated that
the binding between matrine and trypsin was stronger than oxymatrine [33]. The
result is consistent with the result obtained in our previous study using original
enzyme.
The experimental results in Table 2 and Figure (8, 9) indicate that the frontal
affinity chromatography column prepared by the different enzymes with no change in
the primary structure can carry out normal compound affinity screening. Although the
target enzyme inactivation is severe and only 2% of the original enzyme activity, the

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prepared column can still perform affinity screening and obtain effective screening
results.
4. Conclusion
Primary structure of trypsin was unchanged during thermal denaturation, while
its activity decreased from 19555.6 U/g to 222.2 U/g, for the preparation of frontal
affinity chromatography columns. According to preliminary study, small molecule
inhibitors (Oxymatrine and Matrine) of trypsin were selected as analytes, and affinity
screening was performed on seven chromatographic columns prepared by different
activities of trypsins. Compared with the Kd value of original enzyme, other six
devitalized enzymes changed slightly. It is concluded that the binding between
oxymatrine and matrine with trypsin is not affected by the changes of enzyme activity
in frontal affinity chromatographic column.
In the case of serious inactivation of target enzyme (less than 2% of the original
enzyme activity), affinity column is still able to complete the normal screening and
obtain effective results. Thus, affinity columns prepared by different activities of
enzymes can realize the screening effectively of bioactive compounds, as long as
primary structure of target enzyme remains unchanged. It is concluded that the
activity of target enzyme with primary structure unchanged has no effect on bioactive
components screening via FAC. This study provides a basis for genetic engineering to
prepare target enzymes for screening drugs for frontal affinity chromatography.

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Chromatography with Mass Spectrometric Detection: A Method for Finding
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Table 1 Activities of trypsin treated under different conditions
T/℃
20min
30min
A/U/g
40min
50
60
70
13688.9±2.5
2488.9±2.0
444.4±2.8
9022.2±1.5
666.7±1.8
222.2±2.2
6866.7±1.0
622.2±2.0
220.2±2.1
(which T represents temperature; A represents activity of target enzyme)

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Table 2 Determination of Bt for the affinity column and Kd for Oxymatrine and
Matrine at different activity of trypsin
Enzyme
Activity
(U/g)
1
K
d


A

0
Bt
(nmol)
Kd
(μM)

Sample
V V
0
B
t
B
t
Oxymatrine
Y=4.2121x+23.633
0.237
0.211
0.258
0.181
0.294
0.195
0.230
5.600
3.890
5.490
3.209
5.708
3.705
5.257
19555.6
13688.9
9022.2
Matrine
Oxymatrine
Matrine
Y=4.7495x+18.48
Y=3.8644x+21.23
Y=5.5379x+17.772
Y=3.3965x+19.388
Y=5.1024x+18.902
Y=4.3451x+22.846
Oxymatrine
Matrine
Oxymatrine
6866.7
2488.9
Matrine
Y=4.201x+13.338
Y=3.1046x+17.602
0.238
0.322
3.175
5.669
Oxymatrine

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Matrine
Oxymatrine
Matrine
Y=4.4762x+17.293
Y=3.8822x+22.254
Y=4.1642x+14.842
0.223
0.257
0.240
3.860
5.732
3.564
444.4
222.2
Oxymatrine
Matrine
Y=3.6521x+20.002
Y=3.7615x+13.376
0.273
0.266
5.476
3.556