Abstract -Aldo-keto reductase (AKR) was purified to homogeneity from camel liver by ion exchange on Q Sepharose, affinity chromatography on Blue-Sepharose and 2,5-ADP-Sepharose 4B. The purification procedure resulted in 32.43-fold purification with 0.65% final yield. Subunit and native molecular weights of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, were 33kD and 133kD, respectively. The purified AKR exhibited maximal activity at a temperature of 50°C and pH of 7.0. The K m values for NADPH and NADH calculated from the Lineweaver-Burk plot were 0.01 mM and 0.083 mM, respectively, whereas the Km values for m-Nitrobenzaldehyde, 4-Anisaldehyde and P-Benzoquinone were 0.9 mM, 1.11 mM and 0.57 mM, respectively

Abdurrahman M Al-Senaidy

Ibrahim A Al-Harbi

Mohammad A Ismael

CiteSeerX

Arch. Biol. Sci., Belgrade, 65 (1), 113-119, 2013 DOI:10.2298/ABS1301113A
113
PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE 
FROM CAMEL (CaMelus DRoMeDaRius) LIVER
IBRAHIM A. AL-HARBI, ABDURRAHMAN M. AL-SENAIDY and MOHAMMAD A. ISMAEL
Protein Research Chair, Biochemistry Department, College of Science, King Saud University, Riyadh,  
Kingdom of Saudi Arabia
Abstract - Aldo-keto reductase (AKR) was purified to homogeneity from camel liver by ion exchange on Q Sepharose, af-
finity chromatography on Blue-Sepharose and 2,5-ADP-Sepharose 4B. The purification procedure resulted in 32.43-fold 
purification with 0.65% final yield. Subunit and native molecular weights of the purified enzyme determined by sodium 
dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, were 33kD and 133kD, respectively. 
The purified AKR exhibited maximal activity at a temperature of 50°C and pH of 7.0. The Km values for NADPH and 
NADH calculated from the Lineweaver-Burk plot were 0.01 mM and 0.083 mM, respectively, whereas the Km values for 
m-Nitrobenzaldehyde, 4-Anisaldehyde and P-Benzoquinone were 0.9 mM, 1.11 mM and 0.57 mM, respectively.
Key words: Camel, purification, aldo-keto reductase, NAD(P)H, characterization, m-Nitrobenzaldehyde, 4-Anisaldehyde, 
p-Benzoquinone.
INTRODUCTION
Carbonyl reductase (CR) (EC 1.1.1.184) comprises a 
large family of cytosolic NAD(P)H-dependent oxi-
doreductase which catalyzes the reduction of a vari-
ety of carbonyl compounds present in foods and en-
vironmental pollutants. CRs are considered as phase 
I drug metabolizing enzymes for a variety of carbo-
nyl-containing drugs, thereby play a central role in 
xenobiotic metabolism leading to bioactivation or 
detoxication of reactive compounds derived from li-
pid peroxidation and glycation (Yi and Trevor, 2007). 
Other CR endogenous carbonyls include monosac-
charides, steroids, neurotransmitter and prostaglan-
dins (Vincent et al., 2000).  
The characterized carbonyl-reducing enzymes 
are grouped into two distinct protein superfamilies, 
the short-chain dehydrogenases/reductases (SDR) 
aldo-keto reductases (AKR), or medium chain dehy-
drogenases/reductases (MDR) (Oppermann, 2007). 
Mammalian tissues produce enzymatic systems be-
longing to the SDR family or members of the AKR 
family such as aldose reductase, aldehyde reductase, 
dihydrodiol dehydrogenases, and several other re-
ductases. AKR enzymes act on aldehydes and ketones, 
where aldehydes are converted to primary alcohols 
and ketones are reduced to corresponding second-
ary alcohols (Annerita et al., 1995; Wermuth, 1985; 
Matsumoto et al., 2006) that are more hydrophilic 
and can be conjugated and excreted easily. Reac-
tive oxygen species (ROS) production is enhanced 
during the normal aerobic metabolic process. Heat 
stress stimulates excessive production of free radi-
cals that are capable of initiating lipid peroxidation, 
leading to a variety of carbonyl compounds, such 
as malondialdehyde (MDA) and 4-hydroxynonenal 
(HNE) (Bernabucchi et al., 2002).
114 IBRAHIM A. AL-HARBI ET AL.
The Arabian camel (Camelus dromedarius) is 
able to survive in extremely harsh desert conditions 
characterized by a hot and dry summer and extreme-
ly cold winter seasons. The camel is well adapted to 
dehydration for relatively long periods (Souilem O., 
and Barhoumi K., 2009). Metabolic processes in cam-
els may lead to the accumulation of harmful endog-
enous carbonyl compounds and these compounds 
can in turn damage healthy cells by forming adduct 
with proteins and nucleotides. To our knowledge, 
studies describing AKR from Arabian camel liver 
are not available. Therefore, in the present work, the 
purification and biochemical properties of a novel, 
thermostable AKR from Arabian camel are reported 
for the first time. 
MATERIALS AND METHODS
Chromatographic media and molecular weight mark-
ers were from GE Healthcare. Coenzymes, (NADPH 
and NADH) and EDTA was purchased from Sigma 
Chemical Co. (USA). All other chemicals were of 
analytical grade obtained from BDH Chemicals or 
Merck AG (Germany).
Purification of camel liver carbonyl reductase
Camel liver from a local slaughterhouse was trans-
ported to the laboratory on ice. The liver was ho-
mogenized by blender in 20 mM Tris-HCl buffer 
pH 8.0 (buffer A) containing 1 mM EDTA and 10 
mM PMSF and then centrifuged at 9000g for 20 
min. The cold supernatant obtained was loaded 
onto a Q Sepharose column (10 x 2.6 cm) previously 
equilibrated with buffer A containing 1 mM EDTA. 
The column was eluted first with two bed volumes 
of the equilibration buffer, followed by linear gradi-
ent of 0-500 mM NaCl in buffer A at a flow rate of 
2 ml min-1. The fractions were collected using an 
automated liquid chromatography system (ÄKTA 
GE Healthcare). Fractions showing carbonyl re-
ductase activity were pooled and concentrated by 
an Amicon ultra-filtration cell fitted with a PM-10 
membrane (Millipore). CR was further purified us-
ing an affinity column (15 x 1 cm) packed with Blue 
Sepharose. The column was pre-equilibrated with 
50 mM phosphate buffer pH 7.2 (buffer B), and CR 
was eluted by 1M NaCl in the buffer at a flow rate of 
2 ml min-1. Active fractions eluted as a single peak 
were pooled, concentrated to 3 ml and dialyzed 
against buffer B. The dialyzed fraction was applied 
to 2,5-ADP Sepharose affinity column (5 x 1 cm) 
equilibrated with buffer B. The column was washed 
with buffer and CR was eluted by linear gradient of 
0-1 mM NADPH in buffer B. The active fractions 
were pooled and used as the purified enzyme. Un-
less otherwise noted, all steps of enzyme purifica-
tion were carried out at 4°C.
Protein Estimation
Protein concentration was determined by the method 
of Bradford using bovine serum albumin as standard 
(Bradford, 1976). Absorbance at 280 nm was used to 
monitor the protein content in column fractions.
Determination of molecular weight
Subunit molecular weight was determined by sodium 
dodecyl sulfate polyacrylamide gel electrophoresis 
(SDS-PAGE) performed with a Bio-Rad mini-protean 
II electrophoresis unit according to Laemmli (1970) 
in a vertical slap gel apparatus using 12% separating 
and 4% stacking gel with 0.3% cross-linking. The 
gel was stained with Coomassie blue R-250. Protein 
samples were treated with sample buffer containing 
1% SDS and 5% 2-mercaptoethanol at 95°C for 5 min 
prior to electrophoresis. Phosphorylase b (94 kDa), 
bovine serum albumin (BSA) (67 kDa), ovalbumin 
(43kDa), carbonic anhydrase (30 kDa), trypsin in-
hibitor (20.1 kDa) and α-lactalbumin (14.4 kDa) 
were used as standard molecular weight markers.
The molecular mass of the native enzyme was 
estimated by gel filtration through a column that 
had previously been calibrated with the following 
standard protein markers: aldolase (158 kDa), co-
nalbumin (75 kDa), ovalbumin (43 kDa) carbonic 
anhydrase (29 kDa) and ribonuclease (13.7 kDa). 
CR was eluted with potassium phosphate buffer (20 
mM, pH 7.2), containing 0.1M NaCl at a flow rate of 
1 ml min-1. 
PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE  FROM CAMEL LIVER 115
Enzyme assay
CR activity was determined spectrophotometrically 
by monitoring the NADPH oxidation at 340 nm. The 
enzyme was assayed routinely at room temperature 
using a reaction mixture containing 50 mM potas-
sium phosphate buffer (pH 7.2), 0.1 mM NADPH, 
and 1 mM m-Nitrobenzaldehyde in a final volume of 
1.0 ml. The reaction was initiated by the addition of 
an appropriate volume of enzyme solution. One unit 
(U) of activity was defined as the amount of enzyme 
required to oxidize one mole of NADPH per minute 
under the assay conditions. Specific activity was cal-
culated as activity units per mg of protein.
Fig. 1. Elution profile of Q Sepharose fast flow ion exchange 
chromatography observed during purification of camel liver car-
bonyl reductase.
Fig. 2. (A) SDS-polyacrylamide gel electrophoresis (12%) of the 
purified CR from camel liver. Lane 1 shows, purified CR (0.5 μg 
protein) in final step and lane 2, contained a set of marker pro-
teins along with their corresponding molecular masses.
Fig. 3. Effect of pH on the activity of camel liver carbonyl re-
ductase.
Fig. 4. Effect of temperature on the activity of camel liver carbo-
nyl reductase; the enzyme activity was assayed by incubating the 
reaction mixtures at 25-80°C.
Temperature (0C)
116 IBRAHIM A. AL-HARBI ET AL.
Effect of cations on CR activity
The effect of divalent metal ions on CR activity was 
examined by preincubating CR for 15 min at room 
temperature with the cations to be tested (Ba2+, Ca2+, 
Cu2+, Mg2+, Mn2+, Zn2+, Co2+) in 50 mM Tris-HCl 
buffer (pH 7.2) at 0.1 mM final concentration. The 
residual activity of CR was determined by the assay 
procedure presented above.
RESULTS AND DISCUSSION
Enzyme purification
CR was purified from camel liver by three steps, 
anion exchange on Q Sepharose, Blue-Sepharose af-
finity and ADP-Sepharose-4B affinity column chro-
matography. A typical elution profile of CR on Q 
Sepharose column chromatography is shown in Fig. 
1. The scheme utilized resulted in almost 32.43-fold 
purification of CR with a recovery of 0.65%, and the 
final specific activity of the purified enzyme was 1.2 
units per milligram of protein (Table 1). The bind-
ing of the enzyme to Blue Sepharose was found to 
be very tight and high ionic strength was required to 
liberate the enzyme, therefore the enzyme was eluted 
by 1 M NaCl. The purity of the enzyme was shown 
by SDS-PAGE and a single band with a molecular 
weight of 33 kDa was observed (Fig. 2). This molecu-
lar weight is in the range of CR subunits from other 
mammalian sources, including dog liver (Endo et al., 
(A)
(C)
(B)
Fig. 5. Substrate saturation curve and Lineweaver-Burk plot of 
CR activity in presence of different concentrations of substrate 
(A) for p-benzoquinone, (B) for m-nitrobenzaldehyde and (C) 
for 4-anisaldehyde, using NADPH coenzyme at constant con-
centration. The Lineweaver-Burk plot (inset Fig. 5A, B and C) 
was generated by plotting 1/velocity vs. 1/substrate.
PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE  FROM CAMEL LIVER 117
2007), guinea pig liver (Usui et al., 1984), and rabbit 
heart (Imamura et al., 1999). 
Native molecular mass as determined by gel fil-
tration through HiLoad Superdex-200 (16/60) was 
approximately 133 kDa. Thus, the active purified 
camel liver CR is homotetramer.
Optimum pH and temperature
The purified camel liver CR, assayed at different pH 
ranging from pH 4.0 to 9.0, exhibited an optimum 
pH over a broad range between pH 6.0 and 8.0 with 
maximum activity around pH 7.0 (Fig. 3). This opti-
mum pH is similar to human liver CR (Doorn et al., 
2004). When camel liver CR was assayed at different 
temperatures, ranging between 25-80°C, it exhibit-
ed maximum activity at 50°C (Fig. 4). Studies have 
reported a wide range of optimum temperature for 
CRs from different sources. In comparison, CR from 
human liver and brain showed maximum activity at 
37°C and 25°C, respectively (Doorn, 2004 et al; Wer-
muth, 1981), at 30°C for rabbit liver (Imamura et al., 
2003) and 55°C for Candida magnoliae (Wada et al., 
1998). The purified CR was stable when stored in 20 
mM potassium phosphate buffer (pH 7.2) at 4°C and 
-20°C for a period of at least two months and was 
also stable for one week at room temperature.
Kinetic studies
The purified camel liver CR showed activity towards 
many substrates, including p-Benzoquinone, m-Ni-
trobenzaldehyde and 4-Anisaldehyde. The apparent 
Km and Vmax values of CR for reduction of p-Ben-
zoquinone as calculated from the double reciprocal 
plot (Fig. 5A) were 0.57 mM, and 10 U/mg (Table 2), 
respectively. The Km and Vmax values of the enzyme 
for reduction of m-Nitrobenzaldehyde (Fig. 5B) were 
0.90 mM and 4.54 U/mg (Table 2), respectively. The 
Km and Vmax values of the enzyme for reduction of 
4-Anisaldehyde were 1.11 mM and 5.26 U/mg (Fig. 
5C), respectively. The Km value for p-Benzoquinone 
is lower in comparison with m-Nitrobenzaldehyde 
and 4-Anisaldehyde, so the enzyme has a higher af-
finity for p-Benzoquinone compared to the others. 
Fig. 6. Coenzyme saturation curve and Lineweaver–Burk plot of CR activity in presence of different concentrations of coenzyme (A) for 
NADPH and (B) for NADH, using m-nitrobenzaldehyde substrate at constant concentration. The Lineweaver-Burk plot (inset Fig. 6A 
and B) was generated by plotting 1/velocity vs 1/substrate.
(A) (B)
118 IBRAHIM A. AL-HARBI ET AL.
In addition, the catalytic efficiency Kcat/Km is higher 
for p-benzoquinone about 40.4 M-1S-1 (Table 2). 
The camel liver CR can utilize both NADPH and 
NADH coenzymes. The apparent Km and Vmax values 
of CR for oxidation of NADPH as calculated from 
the double reciprocal plot (Fig. 6A) were 0.010 mM, 
and 4.54 U/mg (Table 2), respectively, which is simi-
lar to that reported for CR from human brain (Wer-
muth, 1981). The Km and Vmax values of the enzyme 
for oxidation of NADH (Fig. 6B) were 0.083 mM 
and 4 U/mg (Table 2), respectively. The camel liver 
CR enzyme showed higher affinity for NADPH than 
NADH, so the camel liver has an efficient mechanism 
against toxic and carcinogenic agents.
Effect of metal ions on CR activity
The effect of metal ions on CR activity is presented in 
Table 3. The enzyme activity was enhanced by Ca2+ 
and Ba2+ at 0.1 mM. On the other hand, Cu2+ ions 
strongly inhibited the enzyme activity, as reported 
for CR from other organisms (Kataoka et al., 1992; 
Yamamoto et al., 2002; Nishinaka et al., 1993). The 
enzyme activity was inhibited by Co2+, Zn2+ and Mg2+ 
ions at 0.1 mM. An inhibitory effect of Zn2+ was re-
ported for CR from other organisms (Yamamoto et 
al., 2002; Kizaki et al, 2005). No change in camel liver 
CR activity was noticed with Mn2+ ions. In conclu-
sion, the properties of camel liver carbonyl reduct-
ase appear to be unique because of its good stability, 
wide pH range, wide substrate specificity and use of 
both NADPH and NADH coenzymes.
Acknowledgments - This work was supported by the Protein 
Research Chair, Department of Biochemistry, College of Sci-
ence, King Saud University. 
REFERENCES
Annerita, K., Carina, V.Z., Andrew, V.T., and A. Bernard (1995) 
Prior Purification and Partial Characterization of an Al-
do-Keto Reductase from Saccharomyces cerevisiae. App. 
Env. Microb 61, 1580-1585.
Bradford, M.M., (1976) A rapid and sensitive method for the 
quantitation of microgram quantities of protein utilizing 
the principle of protein-dye binding. Anal. Biochem. 72, 
248-254.
Doorn, J.A., Maser, E., Blum, A., Claffey, D.J., and D.R. Petersen 
(2004) Human carbonyl reductase catalyzes reduction of 
4-oxonon-2-enal. Biochem, 19, 43(41), 13106-14.
Ellis, E.M., and J.D. Hayes (1995) Substrate specificity of an afla-
toxin-metabolising aldehyde reductase. Biochem J., 312, 
535-541. 
Endo, S., Matsunaga, T., Nagano, M., Abe, H., Ishikura, S., Imam-
ura, Y., and A. Hara. (2007) Characterization of an oli-
gomeric carbonyl reductase of dog liver: its identity with 
peroxisomal tetrameric carbonyl reductase. Biol Pharm 
Bull., 30, 1787-91.
Imamura, Y., Koga, T., Shimada, H., and M. Otagiri (2003) Inac-
tivation of rabbit liver carbonyl reductase by phenylgly-
oxal and 2,3,4-trinitrobenzenesulfonate sodium. J Enzyme 
Inhib Med Chem, 18, 35-9.
Imamura, Y., Migita, T., Otagiri, M., Choshi, T., and S. Hibino 
(1999). Purification and catalytic properties of a tetramer-
ic carbonyl reductase from rabbit heart. J. Biochem, 125, 
41-7.
Kataoka, M., Doi, Y., Sim, T.S., Shimizu, S., and H. Yamada 
(1992) A novel NADPH-dependent carbonyl reductase 
of Candida macedoniensis: purification and characteriza-
tion. Arch Biochem Biophys, 294, 469-74.
Kizaki, N., Sawa, I., Yano, M., Yasohara, Y., and J. Hasegawa 
(2005). Purification and characterization of a yeast carbo-
nyl reductase for synthesis of optically active (R)-styrene 
oxide derivatives. Biosci Biotechnol Biochem 69, 79-86.
Laemmli, UK. (1970) Cleavage of structural proteins during the 
assembly of the head of bacteriophage T4. Nature 227, 
680-685.
Matsumoto, K., Endo, S., Ishikura, S., Matsunaga, T., Tajima, K., 
El-Kabbani, O., and A. Hara (2006) Enzymatic properties 
of a member (AKR1C2O) of the Aldo-Keto Reductase 
family. Biol Pharm Bull 29, 539-542.
Nishinaka, T., Kinoshita, Y., Terada, N., Terada, T., Mizoguchi, 
T., and T. Nishihara (1993) Characterization of multiple 
forms of carbonyl reductase from chicken liver. Enzyme 
46, 221-228.
Oppermann U. (2007) Carbonyl reductases: the complex rela-
tionships of mammalian carbonyl- and quinone-reducing 
enzymes and their role in physiology. Annu Rev Pharma-
col Toxicol 47, 293-322.
Souilem O., and K. Barhoumi  (2009) Physiological Particularities 
of Dromedary (Camelus dromedarius) and Experimental 
Implications. Scand. J. Lab. Anim. Sci. 36, 19-29.
PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE  FROM CAMEL LIVER 119
Usui, S., Hara, A., Nakayama, T., and H. Sawada (1984) Purifi-
cation and characterization of two forms of microsomal 
carbonyl reductase in guinea pig liver. Biochem J. 223, 
697-705.
Vincent, P.K., Ireland, L.S., Elizabeth, M.E., and J. Hayes (2000) 
Purification from rat liver of a novel constitutively ex-
pressed member of the aldo-keto reductase 7 family that is 
widely distributed in extrahepatic tissues. Biochem J. 348, 
389-400.
Wada, M., Kataoka, M., Kawabata, H., Yasohara, Y., Kizaki, N., 
Hasegawa, J., and S. Shimizu (1998) Purification and char-
acterization of NADPH-dependent carbonyl reductase, 
involved in stereoselective reduction of ethyl 4-chloro-3-
oxobutanoate, from Candida magnoliae. Biosci Biotechnol 
Briochem 62, 280-5.
Wermuth, B., (1981) Purification and properties of an NADPH-
dependent carbonyl reductase from human brain. Rela-
tionship to prostaglandin 9-ketoreductase and xenobiotic 
ketone reductase. J Biol Chem 256, 1206-13.
Wermuth, B., (1985) Enzymology of carbonyl metabolism 2: 
aldehyde dehydrogenase, aldo-keto reductase, and alcohol 
dehydrogenase. (Alan R. Liss, Inc.), 209-230. New York.
Yamamoto, H., Kimoto, N., Matsuyama, A., and Y. Kobayashi 
(2002) Purification and properties of a carbonyl reductase 
useful for production of ethyl (S)-4-chloro-3-hydroxybu-
tanoate from Kluyveromyces lactis. Biosci Biotechnol Bio-
chem 66, 1775-8.
Yi, J., and T. Pennin, (2007) Aldo-Keto Reductases and Bioacti-
vation/Detoxication. Ann. Rev. Pharm Toxic 47, 263-292.


PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE FROM CAMEL (CaMelus DRoMeDaRius) LIVER

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PURIFICATION AND CHARACTERIZATION OF HIGHLY STABLE ALDO-KETO REDUCTASE FROM CAMEL (CaMelus DRoMeDaRius) LIVER

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