Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique

Article abstract of DOI:10.1016/j.jchromb.2019.03.035

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide d

Full text of DOI:10.1016/j.jchromb.2019.03.035

Accepted Manuscript
Purification of peroxidase enzyme from radish species in fast and
high yield with affinity chromatography technique
Aykut Oztekin, Zuleyha Almaz, Serpil Gerni, Deniz Erel, Seyma
Mehtap Kocak, Mehmet Emin Sengül, Hasan Ozdemir
PII:
S1570-0232(18)31855-5
DOI:
Reference:
https://doi.org/10.1016/j.jchromb.2019.03.035
CHROMB 21578
To appear in:
Journal of Chromatography B
Received date:
Revised date:
Accepted date:
14 December 2018
22 March 2019
26 March 2019
Please cite this article as: A. Oztekin, Z. Almaz, S. Gerni, et al., Purification of peroxidase
enzyme from radish species in fast and high yield with affinity chromatography technique,
Journal of Chromatography B, https://doi.org/10.1016/j.jchromb.2019.03.035
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ACCEPTED MANUSCRIPT
Purification of Peroxidase Enzyme from Radish Species in Fast and High
Yield with Affinity Chromatography Technique
Aykut Oztekina, Zuleyha Almazb, Serpil Gernic, Deniz Erelc,
Seyma Mehtap Kocakc, Mehmet Emin Sengülc, Hasan Ozdemirc
aDepartment of Medical Services and Techniques, Agri Ibrahim Cecen University Vocational
School of Health Services, 04100-Agri, Turkey
bMuş Alparslan University, Faculty of Science Literature, Molecular Biology and Genetics
Department, 49250-Muş
cAtatürk University, Science Faculty, Chemistry Department, 25240-Erzurum
*Corresponding author:
Phone
Fax
: +904422314437
: +904422360948
E-mail
: hozdemir@atauni.edu.tr
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Abstract
In this study, an effective single step affinity method is presented for purifying plant
peroxidase (POD) enzymes from radish species. This method make possible to purify the
enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives
were synthesized and identified as new competitive POD inhibitors. Then, these derivatives
were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity
gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold,
22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD),
black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-
POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-
amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to
purify all radish species POD enzymes in high purity and yield.
Keywords: Peroxidase, Purification, Radish, Affinity chromatography
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1. Introduction
Peroxidases (POD, EC 1.11.1.7) are haeme containing enzyme groups that widely found in
bacteria, fungi, plants and animals. Peroxidases are divided into 3 classes according to amino
acid sequence similarities and metal-ion binding capacities (class I, prokaryotic peroxidases;
class II, extracellular fungal peroxidases; and class III, plant secretory peroxidases) [1]. They
have important roles in physiological processes in plants, such as protecting cells against
oxidative stress, lignin and suberin formation, crosslinking of cell wall components, healing
of wound, defense against pathogens or insects [2-5]. PODs have generally been used in
clinical immunoassays, biochemical detection of glucose, uric acid and cholesterol, treatment
of phenolic contaminations in wastewater and paper industries [6,7]. POD was firstly
characterized from horseradish roots [8], then, different PODs were isolated, purified and
characterized from many plant sources including turnip, radish, soybean, potato, beetroot,
vanilla bean, apple and orange seeds, royal palm, tomato, date palm, black gram, olives,
strawberries and bitter gourd [9-12]. For many years, horseradish plant has been used as the
only commercial source for the production of plant PODs; however, POD can also be
obtained from other radish species. The different PODs can be as efficient as HRP in terms of
stability, yield and substrate specificity [13]. Therefore, improvements of existing procedures
or developing new methods are commercially important.
There are many methods for purification of the PODs in literature. Ammonium sulfate
precipitation [14], ion exchange chromatography [14, 15], affinity chromatography [16] and
two-phase extraction coupled with gel filtration chromatography [17] are some of them.
During the purification of PODs have been encountered with problems such as the
multiplicity of the chromatographic methods, the increase of the cost, the low yield, and the
poorly obtained enzyme when traditional methods are used. Purification of
biomacromolecules including enzymes by affinity chromatography provides advantages over
other chromatographic methods such as yield, purity and efficacy. Reversible inhibitors of
peroxidase (RIP) are important molecules for affinity chromatography. RIP’s can bind the
POD and they contribute separation of the PODs. Some molecules including hydrazines and
azides are known to be potent peroxidases inhibitors (PODIs) [18, 19]. These molecules were
used for purification of PODs from turnip and radish [20].
In this study, new RIP’s (10 different 4-aminobenzohidrazid derivatives) were determined
and Sepharose-4B-L-Tyrosine affinity gels synthesized using the RIP molecules. According
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to our results, use of the new RIP molecules allowed to single step purification of PODs from
black radish, daikon, sweet radish, kohlrabi radish and turnip in high yield and purity.
2. Material and Methods
2.1. Material
Fresh radish spices were purchased from local shops. Methyl 4-aminobenzoate derivatives
obtained from fluorochem ltd U.K., CNBr-activated-Sepharose-4B and L-tyrosine were
purchased from Sigma–Aldrich Co. Prestained Protein Ladder (Thermo Scientific 26616) and
all other chemicals used are analytical grade purchased from sigma Aldrich or Merck.
2.2. Synthesis of 4-amino benzohydrazide derivative compounds
4-amino benzohydrazide derivates (1a-10a) were synthesized from methyl 4-amino benzoate
molecules (1-10). Synthesis mechanisms were presented in Figure 1. 3 mmol of 4-amino
benzoate molecules (1-10) and 2 ml of 64% hydrazine monohydrate were added to 30 ml of
absolute ethanol, and then the mixture was refluxed for 24 hours. After evaporation, the
residue was added to ethanol and was heated-cooled to obtain crude crystals. The crude
crystals were recrystallized from ethanol [21]. Synthesized molecules confirmed by 400 MHz
1H-NMR (Bruker) and Q-TOF LC/MS (Agilent 6530 Accurate Mass).
1
4-Amino 2-methyl benzohydrazide (1a) - H-NMR (400 MHz, DMSO-d6): 9.00 (bs, 1H),
7.04 (d, J = 8.2 Hz, 1H), 6.34 (d, J = 2.1 Hz, 1H) 6.31 (dd, J = 8.2 Hz, J = 2.1 Hz, 1H), 5.29
(s, 2H), 4.25 (bs, 2H), 2.22 (s, 3H). HRMS (TOF MS ES+): m/z calcd for C8H12N3O+ [M+H]+
166.0975, found 166.0971.
4-Amino 2-methoxy benzohydrazide (2a) - 1H-NMR (400 MHz, DMSO-d6): 8.72 (bs, 1H),
7.57(d, J = 8.4Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H) 6.17 (dd, J = 8.4 Hz, J = 1.9 Hz, 1H), 5.68
+
(bs, 2H), 4.37 (bs, 2H), 3.78 (s, 3H). HRMS (TOF MS ES+): m/z calcd for C8H11N3O2
[M+H]+ 182.0924, found 182.0924.
4-Amino 2-nitro benzohydrazide (3a) - 1H-NMR (400 MHz, DMSO-d6): 9.47 (bs, 1H), 7.22
(d, J = 8.4 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 6.72 (dd, J = 8.4, 2.2 Hz, 1H), 6.07 (bs, 2H),
+
4.32 (bs, 2H). HRMS (TOF MS ES+): m/z calcd for C7H9N4O3 [M+H]+ 197.0669, found
197.0672.
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1
4-Amino 2-chloro benzohydrazide (4a) - H-NMR (400 MHz, DMSO-d6): 9.19 (t, J=4 Hz,
1H), 7.08 (d, J=8.4 Hz, 1H), 6.57 (d, J=2.4 Hz, 1H), 6.46 (dd, J=8.4 Hz, J=2.4 Hz, 1H); 5.69
(s, 2H); 4.34 (d, J=4 Hz, 2H). HRMS (TOF MS ES+): m/z calcd for C7H9ClN3O+ [M+H]+
186.0429, found 186.0427.
1
4-Amino 2-bromo benzohydrazide (5a) - H-NMR (400 MHz, DMSO-d6): 9.20 (s, 1H);
7.04 (d, J =8 Hz, 1H); 6.77 (d, J =2 Hz, 1H); 6.50 (dd, J=8 Hz, J=2 Hz, 1H); 5.65 (s, 2H); 4.33
(s, 2H). HRMS (TOF MS ES+): m/z calcd for C7H9BrN3O+ [M+H]+ 229.9924, found
229.9926.
1
4-Amino 3-methyl benzohydrazide (6a)- H-NMR (400 MHz, DMSO): 9.26 (bs, 1H),
7.47(s, 1H), 7.42 (d, J = 8.3 Hz, 1H ), 6.56 (d, J = 8.3 Hz, 1H ), 5.36 (s, 2H ), 4.29 (s, 2H ),
2.05 (s, 3H). HRMS (TOF MS ES+): m/z calcd for C8H12N3O+ [M+H]+ 166.0975, found
166.0971
4-Amino 3-methoxy benzohydrazide (7a) - 1H-NMR (400 MHz, DMSO-d6): 9.37 (bs, 1H),
7.30 (s, 1H), 7.27 (d, J = 8.1 Hz, 1H), 6.60 (d, J = 8.1 Hz, 1H), 5.24 (s, 2H), 4.33 (s, 2H),
+
3.80 (s, 3H). HRMS (TOF MS ES+): m/z calcd for C8H11N3O2 [M+H]+ 182.0924, found
182.0922.
1
4-Amino 3-nitro benzohydrazide (8a) - H-NMR (400 MHz, DMSO-d6): 9.68 (bs, 1H),
8.49 (s, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.73 (bs, 2H), 6.99 (d, J = 8.9 Hz, 1H), 4.41 (bs, 2H).
+
HRMS (TOF MS ES+): m/z calcd for C7H9N4O3 [M+H]+ 197.0669, found 197.0671.
4-Amino 3-chloro benzohydrazide (9a) - 1H-NMR (400 MHz, DMSOd6): 9.45 (s, 1H); 7.71
(d, J=2 Hz, 1H); 7.53 (dd, J=8 Hz, J=2 Hz, 1H); 6.75 (d, J=8 Hz, 1H); 5.87 (s, 2H); 4.35 (s,
2H). HRMS (TOF MS ES+): m/z calcd for C7H9ClN3O+ [M+H]+ 186.0429, found 186.0427.
1
4-Amino 3-bromo benzohydrazide (10a) - H-NMR (400 MHz, DMSO-d6): 9.43 (bs, 1H),
7.85 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 8.5, 2.0 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 5.80 (bs,
1H), 4.33 (bs, 1H). HRMS (TOF MS ES+): m/z calcd for C7H9BrN3O+ [M+H]+ 229.9924,
found 229.9927.
2.3. Peroxidase activity assay
Peroxidase activities was measured by slight modification of following procedure in all
purification steps, Increase of absorbance at 470 nm during the oxidation of guaiacol to tetra
guaiacol (λmax = 470 nm, ε = 26.6 mM−1 cm−1) was used for determination. The reaction
mixture contained; a final concentration 15 mM guaiacol, 7.5 mM H202, and 0.03 M
phosphate buffer (pH 6.0.). The reaction was initiated by the addition of 2 microliters of
enzyme and measurements were recorded at 470 nm for 60 sec-intervals for 3 min. A unit of
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peroxidase activity was defined as the amount of enzyme catalyzing the oxidation of 1 µmol
of guaiacol in 1 min. [22].
2.4. Determination of Inhibition kinetics
The inhibition effects of synthesized molecules (1a-10a) on BR-POD were measured to
determine the potentials to become a ligand. POD activity was measured in the presence of
different concentrations of synthesized molecules (1a-10a) for defining the IC50 values. Three
different inhibitors and five different Guaiacol concentrations were used for determination of
Ki values. Ki and the inhibition type were calculated from Lineweaver–Burk graphs (1/V-
1/[S]) [23].
2.5. Preparation of affinity gels
Affinity gels were synthesized by according to a published procedure [20] with synthesized
molecules (1a-10a) that have a reversible inhibition effect. L-Tyrosine was used as a spacer
arms for Sepharose 4B, and the molecules (1a-10a) were coupled to the Sepharose-4B-L-
Tyrosine via the ring-bound amine group on these molecules by diazotization [24]. Steps of
affinity gels synthesis were checked with FT-IR.
2.6. Preparation of Enzyme Extracts
Crude extracts of each radish species were prepared to load into affinity columns. For this
purpose, 50 g of small cut radish pieces was crushed with liquid nitrogen and homogenized
into 100 mL of 300 mM phosphate buffer (pH 7.0.) by using a blender. The homogenate was
filtered with Whatman filter paper No. 1 and was centrifuged at 20.000 × g for 15 min.
Supernatant was collected and stored -80 °C for using in further purification steps.
2.7. Purification of Peroxidases
The affinity gels were synthesized using 2 g of CNBr activated Sepharose-4B (Sigma C9142)
and loaded into 1 * 10 cm columns. Total gel volumes were between 6-7 ml and varied
according to the ligand bound. Affinity columns were equilibrated with phosphate buffer (10
mM, pH 6.8). Then, 5-6 ml of homogenates were loaded into the columns and were washed
with 25 mM phosphate buffer (pH 6.8). POD enzymes were eluted with 0.5 M NaClO4/0.1 M
NaCH3COO (pH 6.8). Enzymatically active fragments was collected and dialyzed. Protein
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concentrations in the homogenates and purification steps were determined by the modified
Lowry method [25]. Purification yields were calculated with the following formula:
Y = EA1 / EA2 *100
*Y: Yield, EA1: Homogenate Total Activity (EU), EA2: Purified Enzyme Total Activity (EU)
2.8. SDS-PAGE electrophoresis
Molecular masses of the purified PODs, which isolated from different radish sources, were
analyzed by SDS-PAGE (BIO-RAD Mini Protean Tetra System) with polyacrylamide gel
(12%) and stacking gel (4%). POD’s were detected by the silver staining method and
molecular weights were estimated by prestained protein Ladder (Thermo Scientific 26616)
[26].
2.9. Determination of Columns Binding Capacity
Columns binding capacities were determined for defining the best purification columns, after
purification studies with synthesized affinity columns. The column capacity was measured
according to the following procedure: One ml of synthesized gels (1a, 2a, 3a, 7a, 8a, 9a, 10a
of Sepharose-4B-L-Tyrosine) was poured and transferred to 1x10 cm columns, which
equilibrated with equilibration buffer. Then, the columns were saturated with BR-POD
enzyme and unbound enzyme was washed with wash buffer. After the washing process,
bounded POD enzymes were eluted with the appropriate elution buffer and protein amounts
were calculated. The gels were dried, weighed and the POD binding capacity was determined
as µg protein / g gel.
3. Results
3.1. Inhibition Studies
BR-POD was purified for determining inhibition effects of the synthesized molecules (1a-
10a) according to an existing procedure [20]. POD activities were calculated by adding
different concentrations of inhibitors to the reaction medium at non-saturated guaiacol
concentration. Activity-Concentration (%) graphs were created, and then IC50 values were
calculated. The results are summarised in Table 1. Inhibition types were determined to set
whether or not the synthesized molecules (1a-10a) have potential to be used as ligands. This is
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important for purification process of enzymes because only the reversible-inhibitors can be
used as ligand. Ki values that indicating interest of the enzyme to the inhibitors were also
calculated. Ki values obtained from inhibition studies with BR-POD are presented in Table 1.
It was determined that all molecules could be used as a ligand for purification according to
their Ki values.
3.2. Synthesis of Affinity Gels and The FT-IR Results
Affinity gel was prepared with the molecules showing reversible inhibition. As shown in
Figure 2, 4-amino benzohydrazide derivatives coupled to Sepharose-4B-L-Tyrosine support
matrix by diazotization to form the affinity gels. The binding patterns for Sepharose-4B-L-
Tyrosine-1a gel were determined from the obtained FT-IR results. As shown in Figure 3, the
Sephorase-4B / imidocarbonate structure corresponding to the C-O tension exhibits a weak
peak at 858 cm-1 with a broad and intense band at 1380 cm-1, as expected. In addition, the
-1
spectrum exhibits a very weak band corresponding to the C = N tension at about 1650 cm
(a).
Sepharose-4B exhibits a broad band as a result of L-Tyrosine binding symmetrical /
asymmetric stretches of phenolic C-OH, seconder amine N-H, aromatic C-H and aliphatic C-
H in the range of 3500-2500 cm-1 wavelengths respectively. The low frequency region of the
spectrum confirms that the phenolic ring and carboxylic acid groups are involved in the
-1
structure, 1720, 1640, 1365 and 1050 cm , respectively, corresponding to acidic C = O,
aromatic C = C and phenolic C-OH stretches (b). Observation of new peaks originating from
the N = N and N-NH stretches at 1576, 1429 and 1150 cm-1 wave numbers as well as the
peaks present in the spectrum confirm that the desired coupling of diazonium salt to
sepharose-4B-tyrosine takes place. (c).
3.3. Purification of POD Enzymes by Affinity Chromatography
After the synthesis of affinity gels, homogenates, which amounts of proteins were determined
and enzyme activities were measured, were loaded on affinity columns. When 4-amino 2-
nitro benzohydrazide (3a) were used as ligand, the BR-POD enzyme was purified in 33%
yield and 613-fold and The T-POD enzyme was purified in 54.8% yield and 665-fold. While
Molecular weight of BR-POD was determined as 47 kDa, Molecular weight of T-POD
enzyme was determined as 47 kDa by SDS-PAGE. The SDS-PAGE images are presented in
Figures 4a and 4b. 4-Amino 2-nitro benzohydrazide (3a) was also found to be the most
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effective ligand for purification both of BR-POD and T-POD enzyme. When the 4-amino 3-
bromo benzohydrazide (10a) was used as ligand, the SR-POD enzyme was purified in 34.4%
yield and 781-fold. SDS-PAGE was performed to determine the purity and molecular weights
of the SR-POD enzyme Figure 4b. Molecular weight of SR-POD enzyme was determined as
47 kDa. It was also determined that all 4-amino benzohydrazide derivatives could be used as
ligand in purification of KR-POD enzyme (Table 2). Especially, 4-amino 3-methyl
benzohydrazide (6a) molecule has achieved significant success in purification of the enzyme
and this molecule enabled to purification of the enzyme in 41% yield and 282-fold.
Molecular weight of the enzyme was found to be 47 kDa (Figure 4c). 4-amino 2-methyl
benzohydrazide (1a) and 4-Amino 2-methoxy benzohydrazide (2a) were determined to be
most effective ligands in purification of D-POD enzyme. Purification factors of D-POD
enzyme were recorded as 22.7% yield 595-fold and 17.8% 175-fold, respectively. Molecular
weight of the enzyme was found to be 48.5 kDa (Figure 4d). All purification results are
presented in Table 2 and the detailed purification results of 4-amino 3-bromo benzohydrazide
(10a) are presented in Table 3.
3.4. Column binding capacity studies
Effects of the temperature, pH and ionic strength on column binding capacity were
investigated for BR-POD enzyme. Binding capacity was defined as protein/gram gel. The
column binding capacity of 4-Amino 3-bromo benzohydrazide (10a) was determined as
1353.3 µg/g gel at the conditions 15 °C, pH 7.5 and 0.3 M ionic strength. The optimal
conditions of the ligands for purification of the BR-POD enzyme are presented in Table 4.
4. Discussion
Peroxidases are enzyme families that are used in industry and they have commercial
importance. Peroxidase enzymes are used in many bioprocesses such as cleaning of phenolic
compounds from wastewaters, removing of synthetic dyes, clinical diagnosis and organic
synthesis reactions [1, 27]. Because of the common utilizations, studies on purification of the
peroxidases are increasingly important. Many researchers have been working on purification
of peroxidases by different chromatographic methods. Among these methods, affinity
technique provides superiority to other techniques because it is short-term renewable, one-
step and cheap. In previous studies, POD was purified from papaya, spinach leaves, black
radish, turnip, daikon, orange peels, red cabbage and orange in 30.22 fold with a yield of
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44.37% in three chromatographic steps [28], 22.6-fold with a yield of 22.6 % in two
chromatographic steps [29], 40.3-fold with a yield of 10.6% in single chromatographic step
[20], 269-fold with a yield of 9% in single chromatographic step [20], 60-fold in two
chromatographic steps [30], 18-fold with a yield of 94% in two chromatographic steps [31],
120-fold with a yield of 120% in single chromatographic step [32] and 2.5-fold with a yield of
82.5% in two chromatographic steps [3], respectively.
According to our knowledge, many chromatographic methods and their combinations have
been used in the purification of the peroxidase enzymes. While yields vary ranged from 94%
to 9%, purification folds vary ranged from 2.5 to 269. In the studies that obtained high yield,
the purification fold generally decreased and high purification folds also caused low yields. It
is desired to be high both of purification folds and yields for an effective purification method.
In this study, the peroxidase enzymes from different radish species were purified in yields of
more than 30% and purification folds of more than 500. Single-band was observed in SDS-
PAGE from purified POD enzymes. It was shown that 4-amino benzohydrazide derivative
molecules were only specific to peroxidase enzyme and the molecules did not interact with
other proteins in homogenates.
Structural differences in synthesized derivatives are an important factor effecting enzyme
interaction. Both of the substituent (-CH3, -Cl, -Br, -OCH3, -NO2) and the position to which it
was binding (2 or 3) significantly changed the purification fold and yield. -NO2 and -Br
functional groups bound to the ring increased the efficiency of the purification. (See Table 2,
3a, 10a). -Cl bonded to position 3 decreased the affinity of the ligand for D-POD and reduced
the yield by up to 3%. OCH3 was more effective in positions 2 than position 3. While CH3 on
the position 3 was very effective in the purification of KR-POD, it did not show the same
effect in the purification of other PODs. As shown in Table 2, the effectiveness of purification
is related to the type of functional group, the binding position and the type of peroxidase.
Detailed kinetic studies were conducted for the synthesized molecules (1a-10a) and these
molecules were defined as new inhibitors of the peroxidase enzymes. Additionally, SR-POD
and KR-POD enzymes were purified with this study first time, successfully.
5. Conclusions
In order to purify quickly, cheaply and in high yield the peroxidase enzymes from radish
species, an affinity chromatography technique was developed on the Sepharose-4B-L-tyrosine
carrier matrix by using 4-amino benzohydrazide derivatives as ligands. In particular, it was
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achieved that high yields and purity in purification by using 4-amino 3-bromo and 4-amino 2-
nitro benzohydrazide molecules. This method is superior to existing methods in terms of yield
and purification factor. In addition, it significantly shortens the time spent in obtaining POD
enzymes.
Acknowledgment
This study was supported by TUBITAK with 215Z386 project number.
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Figure 1. Synthesis route of 4-amino benzohydrazide derivatives (1a-10a)
14

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Fig 2. Synthesis of the Sepharose-4B-L-Tyrosine-(1a-10a) affinity gels.
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Fig 3. FTIR (Burker VERTEX 70V) results of a: Sepharose-4B imidocarbonate, b: Sepharose
4B-L-Tyrosine, c: Sepharose 4B-L-Tyrosine-4-Amino 2-methyl benzohydrazide (1a) affinity
gel.
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Fig 4. SDS-PAGE bands of T-POD, BR-POD, D-POD, SR-POD and KR-POD Enzymes
purified from Sepharose 4B-L-Tyrosine-4-amino 3-bromo benzohydrazide affinity jel. M:
Markers (Thermo Scientific 26616). (a); 1: BR-POD, (b); 1: T-POD, 2: SR-POD, (c); 1: KR-
POD, (d); 1: D-POD.
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Table 1. IC50 value, Ki constant and inhibition type of 4-amino benzohydrazide derivatives for
BR- POD.
Inhibitors
IC50 (mM)
Ki (mM)
Inhibition type
0,371
0,415 ± 0,0862
Non-competitive
1a
2a
3a
0,231
0,961
0,271 ± 0,019
1,04 ± 0,0649
Non-competitive
Non-competitive
0,346
0,356 ± 0,055
0,547 ± 0,081
0,00015 ± 0,000032
0,103 ± 0,039
0,356 ± 0,116
0,047 ± 0,017
Non-competitive
Competitive
4a
5a
6a
7a
8a
9a
0,373
0,000124
0,086
Non-competitive
Non-competitive
Non-competitive
Competitive
0,440
0,173
0,115
0,127 ± 0,006
Non-competitive
10a
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Table 2. Purification results of POD enzymes from radish species with Sepharose-4B-L-
tyrosine-(1a-10a) affinity gels.
BR-POD
T-POD
SR-POD
KR-POD
% Yield
13
D-POD
% Yield
22,7
17,8
10
Ligand
% Yield Fold
% Yield Fold
% Yield Fold
Fold
581
262
705
582
424
282
337
745
378
245
Fold
595
175
363
342
130
291
557
371
156
388
14,1
33,6
33,8
14,5
28,5
14,2
10,1
7,5
211
676
613
263
258
191
244
365
248
677
11,7
20,1
54,8
8,1
444
806
665
292
330
234
653
403
676
1348
20,2
30,2
14,9
6
591
322
678
273
601
103
412
502
236
781
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
6,8
21,3
15,3
24
4,3
18,2
10,2
12,2
10,1
13,6
22
26,5
15,8
9,1
16,5
5,91
13
40,9
16,3
21
6,1
4,7
19,5
31,4
4,6
9,9
3
13,8
9,8
34,4
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Table 3. The detailed purification results of Sepharose 4B-L-Tyrosine-4-amino 3-bromo
benzohydrazide affinity gel
SOURCE
STEP
aStep1
bStep2
aStep1
bStep2
aStep1
bStep2
aStep1
5
3
6
3
6
3
6
108
1,45
540
7,25
74,5
100
31,4
100
9,86
100
13,8
100
1
Black radish
Daikon
56,5
39,2
7,74
0,0011 169
0,003
50454
33,2
677
1
1,18
235,2 7,08
0,0006 23,2
0,0018 12900
388,5
1
140,4 1,24
842
7,4
113,2
27785
50,6
Kohlrabi radish
38,9
51,7
0,0014 116
0,004
245,4
1
1,02
310,2 6,12
Sweet radish
Turnip
bStep2
aStep1
bStep2
3
35,6
148
0,0009 106,8 0,0027 39555,5 34,4
781,7
1
5
1,29
740
6,45
114,7
100
22
1,5
108,2 0,0007 162,3 0,001
154571
1348
aStep1: Crude Homogenate
bStep2: Sepharose 4B-L-tyrosine-4-amino benzohydrazide affinity column chromatography
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Table 4. Optimal parameters of affinity gels for the purification of BR-POD enzyme
Loaded
Optimum
Ionic
Binding
capacity
BR-POD
Samples
(mg/g dry
gel)
Optimum
pH
Optimum
Recovery
(%)
Ligand
Temperature
strength
(µg/g dry gel)
8
8
7
7
15
15
20
25
15
10
15
0,3
0,35
0,3
0,4
0,4
0,4
0,3
223,2
1400
4
4
4
4
4
4
4
5,5
1a
2a
3a
7a
8a
9a
10a
35
2660
56,5
50
1916,6
751,06
573,3
1353,3
7,5
7,5
7,5
18,8
14,3
33,8
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Highlights
 10 different 4-amino benzohydrazide derivatives were synthesized and identified as
new POD inhibitors.
 These derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix to form
affinity gels.
 The binding patterns for Sepharose-4B-L-Tyrosine-(1a-10a) affinity gels were
controlled by FT-IR.
 The radish peroxidases were purified in a single step with the prepared affinity gels.
22