Wiley

BioMed Research International

Volume 2024, Article ID 2222098, 8 pages
https://doi.org/10.1155/2024/2222098

Research Article
Single-Step Purification of Catalase Enzyme From Human Blood
Erythrocytes Using Affinity Chromatography Technique

Kübra Çıkrıkcı and Nahit Gencer

Department of Chemistry, Faculty of Arts and Sciences, Balikesir University, Balikesir, Türkiye

Correspondence should be addressed to Nahit Gencer; [email protected]

Received 6 February 2024; Revised 14 May 2024; Accepted 5 June 2024

Academic Editor: Luis Morales-Quintana

Copyright © 2024 Kübra Çıkrıkcı and Nahit Gencer. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.

In this study, we aimed to isolate and purify catalase from human blood erythrocytes by using a newly synthesized affinity gel. The
synthesized ω-amino hexyl agarose-1,2,3-triazole-5-carboxylic acid affinity gel was analyzed by FT-IR. Then, different buffer, pH,
and ionic strength parameters were optimized to determine the equilibration, washing, and elution buffer conditions. The catalase
was purified from human blood erythrocytes with a specific activity of 45.58 EU/mg, purification fold of 529.50, and a yield of
0.416% using the synthesized new affinity gel. The purity and molecular weight of the enzyme were analyzed by SDS-PAGE,
and a single band at 60 kDa was observed for catalase. The optimum reaction temperature of the catalase was found to be
30°C, while the thermal stability temperature was 60°C. The Km and Vmax of the enzyme for hydrogen peroxide were
calculated at 0.125 mM and 2500 U mL−1, respectively.

Keywords: affinity chromatography; catalase; human blood erythrocytes; purification

1. Introduction sues such as the heart and brain. Human erythrocyte catalase
(HEC) is a 244 kDa tetrameric protein with more than 500
Enzymes are environmentally friendly biocatalysts with high amino acid residues in each molecule, each containing four
efficiency that are used in industrial fields for sustainable identical subunits of 59.7 kDa bound to its active site, four
production. As the demand for enzymes increases in indus- heme groups, and four NADPHs [13]. In addition, it has
trial fields, their approach to purification is expected to been reported that cyanide, azide, and fluoride cause
improve [1]. Enzymes can be purified using many different reversible inhibition of catalase, while 3-amino-1,2,4-triazole
chromatographic purification methods. However, in indus- (3-AT) causes irreversible inhibition [14]. Affinity chroma-
trial areas, one-step purification of enzymes from raw tography is a practical alternative for one-step purification
sources is very important to reduce production costs [2, 3]. of enzymes based on the principle of specific immobilization
Catalase is widely used in industry for H2O2 removal, of a biological ligand (for example, substrate, coenzyme, hor-
oxidation prevention, biosensors, and bioremediation mone, and inhibitor) on an insoluble support material
[4–7]. Catalase is also widely used in clinical studies as it is (matrix) [15]. The selectivity and simplicity of this method
an antioxidant enzyme, and catalase deficiency has been are exploited for the purification of many biomolecules, bio-
associated with several diseases, such as acatalasemia, viti- pharmaceuticals, and other agents [2].
ligo, coronary artery disease, cancer, and type 2 diabetes In this study, a novel affinity gel was synthesized and
[8–12]. Catalase (EC 1.11.1.6), an antioxidant enzyme optimized. Then, catalase was purified from human blood
located in the peroxisomes of nearly all aerobic microorgan- erythrocytes. In the literature, catalase has been purified by
isms, plants, and animals, protects cells from ROS by reduc- different chromatography techniques and from different
ing hydrogen peroxide to water and oxygen. Catalase activity sources, but studies on catalase purification from human
is high in mammalian liver and erythrocytes and low in tis- blood erythrocytes are limited. In addition, the affinity
2 BioMed Research International

chromatography technique, a type of adsorption chromatog- equilibration buffer. Then, the wash buffer was prepared
raphy, and 3-AT-5-carboxylic acid ligand were used in this with 25 mM Tris-base/22 mM NaCl at the same pH values
study for the first time. as the equilibration buffer optimization. However, to deter-
mine the ionic strength, 25 mM Tris-Base (pH: 9.5) was pre-
2. Materials and Methods pared with different concentrations (22-24-26-28 mM) of
NaCl, Na2SO4, and NaSCN and the column was washed
2.1. Chemicals. ω-Aminohexyl agarose, hydrogen peroxide with these buffers. Finally, 0.15 M Na2HPO4 (pH:5) was pre-
(H2O2), dipotassium phosphate (K2HPO4), potassium dihydro- pared at different pH (9.5–5) and concentrations (20-24-
gen phosphate (KH2PO4), Tris-HCl, N,N,N’,N’-tetramethyle- 28 mM) with NaCl, Na2SO4, and NaSCN, respectively, to
nediamine (TEMED), tri-hydroxymethylaminomethane determine the ionic strength for the elution buffer. Purifica-
(Tris-Base), 3-AT-5-carboxylic acid, and 1-ethyl-3-(3- tion was carried out with all the prepared buffer solutions
dimethylaminopropyl) carbodiimide hydrochloride (EDC) separately, and the highest activity elution condition for
were obtained from Sigma Chemical Co. Prestained Protein the catalase was determined as 0.2 M Na2HPO4 (pH: 5).
Ladder, “PageRuler™ Plus” 10–180 kDa, was purchased from
Thermo Fisher Scientific. All other chemical materials used 2.2.4. Optimum and Stability Temperature of Catalase. To
are of analytical purity. Human blood samples were obtained determine the optimum temperature at which the activity
from Balikesir University Faculty of Medicine. The study of catalase was purified from human blood erythrocytes, it
was approved by the local Institutional Ethics Committee, was measured spectrophotometrically with 10°C of increases
and informed consent was obtained from all participants. ranging from 10°C to 60°C. These measurements were
repeated three times, and an activity-temperature plot was
2.2. Methods generated. The thermal stability of purified catalase was
determined by incubating it at various temperatures (4°C,
2.2.1. Synthesis of Affinity Gel for Catalase Purification. 20°C, 30°C, 40°C, 50°C, and 60°C) for 1 h, with enzyme activ-
Twenty microliters of ω-aminohexyl agarose was prepared ity measured spectrophotometrically and recorded every
by washing with 60 mL of distilled water. This procedure 10 min.
was repeated three times. Thirty milligrams of 3-AT-5-car-
boxylic acid (ligand) was dissolved in 40 mL of distilled 2.2.5. Protein Determination. Quantitative protein determi-
water. Then, 100 μL of 1-ethyl-3-(3-dimethylaminopropyl) nation was performed at 595 nm by the Bradford method
carbodiimide hydrochloride (EDC) crosslinking reagent [17]. Bovine serum albumin (BSA) was used as a standard,
was added to the ligand solution. The prepared ligand solu- and a purification table was prepared.
tion was rapidly added to the matrix solution. The pH was
kept constant at 4.5 for the reaction to take place, and the 2.2.6. SDS-PAGE Analysis. To check the purity of the purified
solution was incubated at 25°C for 18 h. The gel solution catalase and determine its molecular weight, SDS-PAGE was
was washed with 1 L of distilled water and 250 mL of NaOH performed according to the Laemmli method [18].
(pH: 4.5) buffer by transferring the Buchner funnel. The syn-
thesized ω-aminohexyl agarose-3-AT-5-carboxylic acid 2.2.7. Determination of Catalase Activity and Kinetic
affinity gel was analyzed by FT-IR. This analysis was per- Parameters. The catalase activity was measured spectropho-
formed with a Perkin Elmer Spectrum 100 device in the tometrically at 240 nm according to the method of Aebi [19].
wavelength range of 4000–650 cm−1. One unit (enzyme units [EUs]) of the catalase activity was
defined as the amount of enzyme that catalysed the decom-
2.2.2. Preparation of Hemolysate. Blood sample (10 mL) was position of 1 μmol H2O2 (30 mM) at 25°C in phosphate
collected from healthy volunteers. The blood sample was buffer (50 mM, pH 7.0) in 60 s. The molar absorptivity for
centrifuged at 5000 rpm for 20 min at 4°C. Plasma and buffy H2O2 at 240 nm was assumed to be 40.0 M−1 cm−1. Enzyme
coat were separated and washed twice with 0.9% NaCl. activity was assessed at a wavelength of 240 nm using six dif-
Then, the blood sample was hemolyzed with three times ferent substrate concentrations at pH 7.0 in order to deter-
the volume of cold water. The hemolysate was centrifuged mine Km and Vmax values. Following the measurements,
again at 15,000 rpm for 40 min at 4°C. calculations were conducted by plotting the Lineweaver–
Burk graph (1/[V]-1/[S]). Each activity measurement was
2.2.3. Optimization of Equilibration, Washing, and Elution repeated three times and recorded.
Buffers of the Affinity Gel. For the optimization of equilibra-
tion, washing, and elution buffers of the affinity column, 3. Results
each condition was prepared as described in this section,
and 10 mL of hemolysate was used for each. The equilibra- In this study, catalase was purified from human blood eryth-
tion buffer was prepared with 25 mM Tris-Base/0.1 M NaCl rocytes by affinity gel with ω-aminohexyl agarose-3-AT-5-
in different pH values (9.5-9-8.5-8-7.5-7-7-6.5-6-6-5.5-5) carboxylic acid chemical structure (Figure 1). Commercially
[16]. The column was equilibrated with each buffer individ- purchased ω-aminohexyl agarose gel (matrix) eliminates the
ually, and the optimum pH was determined to be 8.5. Subse- need for a spacer arm because it contains an amino group
quently, 25 mM Tris-Base (pH: 8.5) was prepared with (-NH2) and has a length of six carbons. The high degree of
different concentrations (0.05-0.1-0.15-0.2 M) of NaCl, purification in affinity chromatography gel synthesis
Na2SO4, and NaSCN to determine the ionic strength of the depends on the specificity and degree of binding of the
BioMed Research International 3

CH3
C + N NH
N N NH Cl–
NH2 CH3
O NH CH3
EDC H 2N
N COOH
pH:4.5
N+
�-aminohexyl agarose 3-amino-1,2,4-triazole-5-carboxylic acid

NH2 C CH3
H N +
NH Cl–
CH3 N N
H
O NH CH3
N N O N

N
N+ H
O
NH NH2

�-aminohexyl agarose -3-amino-1,2,4-triazole-5-carboxyclic acid N

Binding of carbodiimide to ligand

Figure 1: Preparation of the new affinity gel for purification of the CAT enzyme.

99,8 (a)
99,5

99,0
(b)
98,5

98,0 3778,5
2394,2
2992,2
97,5 1494,4

2164,4 1976,2
97,0 1205,5
3294 1646,7
96,5
%T

1710,4 1372,6

96,0 821,65

95,5

95,0
929,63
94,5

94,0

93,5
1043,1
93,1
4000 3500 3000 2500 2000 1500 1000 650
cm–1

Figure 2: FT-IR analysis of the aminohexyl agarose gel which was used as a matrix and the synthesized affinity gel was evaluated in the
wavelength range of 4000-650 cm−1. (a) ω-Aminohexyl agarose (red diagram). (b) ω-Aminohexyl agarose-3-amino-1,2,4-triazole-5-
carboxylic acid (blue diagram).
4 BioMed Research International

Equilibration buffers Wash buffers

250 200

200
150
Activity (U)

Activity (U)
150
100
100

50
50

0 0
0.05 0.1 0.15 0.2 0.022 0.024 0.026 0.028
Ionic strength (I) Ionic strength (I)
NaCl NaCl
Na2SO4 Na2SO4
NaSCN NaSCN
(a) (b)
Elution buffers Elution buffers (Na2HPO4)
200 250

200
150
Activity (U)

Activity (U)

150
100
100

50
50

0 0
0.02 0.022 0.024 0.026 0.028 0.05 0.1 0.15 0.2
Ionic strength (I) Ionic strength (I)
NaCl
Na2SO4
NaSCN
(c) (d)

Figure 3: (a) Determination of the ionic strength of the equilibration buffer (25 mM Tris-Base was prepared 0.05-0.1-0.15-0.2 M NaCl,
Na2SO4, and NaSCN, respectively). (b) Determination of the ionic strength of the wash buffer (25 mM Tris-Base was prepared 0.022-0.1-
0.024-0.026-0.028 mM NaCl, Na2SO4, and NaSCN, respectively). (c) Determination of the ionic strength of the elution buffer (25 mM
Tris-Base was prepared 0.02-0.024-0.026-0.028 mM NaCl, Na2SO4, and NaSCN, respectively). (d) Determination of the ionic strength of
the elution buffer (Na2HPO4 at different concentration was prepared as 0.05 M, 0.1 M, 0.15 M, and 0.2 M, respectively).

selected ligand. For this reason, 3-AT-5-carboxylic acid, a trum range 1900–1650 cm−1 [23]. The primary amide group
specific inhibitory derivative of the catalase, was preferred gave a peak at 1710.4 cm−1 in the synthesized affinity gel. A
as the ligand. In order to activate the amino group (-NH2) comparison of the FT-IR spectra analysis results is shown
in the gel structure and the carboxyl group (-COOH) in in Figure 2. As a result of the affinity gel optimization stud-
the ligand structure, affinity gel was synthesized using the ies, it was determined that the most appropriate buffer for
carbodiimide (EDC) activation method. Binding of the equilibration was 25 mM Tris-Base/0.05 M NaCl (pH: 8.5)
ligand to the primary amino group of the matrix through (Figure 3(a)), while the most appropriate buffer for washing
the carbodiimide forms an amide bond, and this bond is was 25 mM Tris-Base/26 mM NaCl (pH: 9.5) (Figure 3(b)).
resistant to chromatographic processes [20, 21]. FT-IR anal- It was determined that the catalase activity was low by ionic
ysis of the aminohexyl agarose gel which was used as a strength assays for elution (Figure 3(c)). Subsequently,
matrix and the synthesized affinity gel was evaluated in the purification was performed using Na2HPO4 (pH: 5) buffers
wavelength range of 4000–650 cm−1. The IR spectra of the prepared at different concentrations (0.05 M, 0.1 M, 0.15 M,
ω-aminohexyl agarose gel showed -NH peaks between and 0.2 M) (Figure 3(d)). It was found that the highest
3378.5 and 3294 cm−1 [22]. Correspondingly, the -NH group activity for elution buffer was obtained with 0.2 M Na2HPO4
shows a peak between 3500 and 3350 cm−1. In addition, IR (pH: 5). The activity variation of equilibration, washing, and
analysis of the primary amide group gives a peak in the spec- elution buffers at different pH values is shown in Figure 4.
BioMed Research International 5

Optimum pH
200

150

Activity (U)
100

50

0
5 5.5 6 6.5 7 7.5 8 8.5 9 9.5

pH

Equilibration buffers
Wash buffers
Elution buffers

Figure 4: Optimization of equilibration, washing, and elution buffers at different pH ranges (9.5–5).

Table 1: Summary of purification procedure for human blood erythrocyte catalase by a ω-aminohexyl agarose-3-amino-1,2,4-triazole-5-
carboxylic acid affinity column chromatography.

Purification Total volume Activity Total activity Protein Total protein Specific activity % Purification
step (mL) (EU/mL) (EU) (mg/mL) (mg) (EU/mg) yield fold
Hemolysate 10 134.25 1342.5 1559.60 15596 0.086 100 —
Catalase 3 186 558 4.081 12.242 45.579 0.416 529.50

Quantitative protein determination was performed by the 180 kDa a

Bradford method for catalase purified from human blood 130 kDa b
erythrocytes by affinity chromatography. The catalase was
purified from human blood erythrocytes with a specific 100 kDa c.
activity of 45.58 EU/mg, purification fold of 529.50, and a
yield of 0.416% using ω-aminohexyl agarose-3-AT-5-car- 70 kDa
d
boxylic acid affinity chromatography (Table 1). To deter-
mine enzyme purity and molecular mass, SDS-PAGE was
performed, and a single band was obtained at a molecular 55 kDa e 60 kDa
weight of approximately 60 kDa (Figure 5). The determined
molecular mass was similar to that of catalase purified by
different chromatographic methods in previous studies. For 40 kDa f
example, dog erythrocytes at 57.5 kDa [24], sheep erythro-
cyte at 60.52 kDa [25], and human erythrocytes at 60 kDa
[26] were reported. The activity of purified catalase at differ-
ent temperatures (10-20-30-40-50-60°C) was assayed and 35 kDa g
.
determined that the optimum reaction temperature was
30°C (Figure 6(a)). The optimum temperature determined
was similar to the catalase purified from different tissues in
previous studies. For example, chicken liver at 30°C [25], 25 kDa h
2
sheep erythrocyte at 30°C [25], camel liver at 45°C [27], 1
and bovine liver at 40°C [28] were reported. Temperature Figure 5: SDS polyacrylamide gel electrophoresis of human blood
stability was tested for 1 h by measuring catalase activity erythrocyte CAT purified by affinity gel. Line 1: the protein
every 10 min at different temperatures. The catalase showed standards ((a) 180 kDa, (b) 130 kDa, (c) 100 kDa, (d) 70 kDa, (e)
a high activity over a wide range of temperatures, from 4°C to 55 kDa, (f) 40 kDa, (g) 35 kDa, and (h) 25 kDa). Line 2: human
30°C, and showed low activity at 40-50-60°C (Figure 6(b)). blood erythrocyte CAT.
6 BioMed Research International

Optimum temperature
200
180
160
140

Activity (U)
120
100
80
60
40
20
0
10 20 30 40 50 60
Temperature (°C)
(a)
Thermal stability
160

140

120

100
Activity (U)

80

60

40

20

0
10 20 30 40 50 60

Time (min.)
4C 40 C
20 C 50 C
30 C 60 C
(b)

Figure 6: (a) The effect of different temperatures on CAT activity. (b) Thermal stability of CAT at different temperatures.

The enzyme kinetics of the catalase were measured spectro-

0.0120
photometrically using different H2O2 concentrations as sub-
strates, and a Lineweaver–Burk plot was used to calculate y = 5E–05x + 0,0004
0.0100 R² = 0,9961
Km: 0.125 mM, and Vmax: 2500 EU/mL (Figure 7).
1/V (�mol/dk)–1

0.0080
4. Discussion
0.0060
Affinity chromatography is a protein purification technique
based on the specific immobilization of a biological ligand 0.0040
(substrate, coenzyme, hormone, antibody, nucleic acid,
0.0020
inhibitor, etc.) on an insoluble support material (matrix)
[15]. Affinity gel synthesis consists of several steps, including 0.0000
immobilization of the ligand to the matrix, removal of –50 0 50 100 150 200 250
unwanted impurities, and pure elution of the target protein. –0.0020
1 (S)
Various gels such as Sephadex, Sepharose, and Biogel are
used as the matrix. The functional groups of the carrier Figure 7: Lineweaver–Burk graph of human blood erythrocyte
matrix (-S, -SH, -OH, -NH3, etc.) are very important for CAT obtained by using H2O2 substrate.
the covalent binding of the groups in the side chains of the
amino acids in the enzyme structure [29, 30]. The matrix
BioMed Research International 7

and ligand must be activated with reagents such as cyanogen Funding

bromide, carbodiimide, and glutaraldehyde for this binding
to occur. After the ligand immobilization is performed to This study has been supported by Balikesir University
the activated matrix, the molecule to be purified is bound (BAUN) Research Projects with project No. 2023/134.
to the ligand on the column. The molecule retained in the
column is eluted with a buffer solution containing a sub-
stance of higher affinity than the ligand, and hundreds of Acknowledgments
fold purification are achieved in a single step [31, 32].
This study has been supported by Balikesir University
In this study, catalase was purified from human blood
(BAUN) Research Projects with project No. 2023/134.
erythrocytes by one-step ω-aminohexyl agarose-3-AT-5-car-
boxylic acid affinity chromatography with 529.60-fold.
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