Comparing inhibitory effect of tramadol on

catalase of Pseudomonas aeruginosa and mouse liver

Running title: Tramadol and catalase inhibition

Dariush Minai-Tehrani*, Imana Sadat Ashrafi, Mina

Kolahdoz Mohammadi, Zahra Sobhani Damavandifar, Elmira

Rezaei Zonouz, Tahmineh Ebrahimzadeh Pirshahed

* Corresponding author: Dariush Minai-Tehrani (PhD)

BioResearch Lab, Faculty of Biological Sciences, Shahid

Beheshti University, G.C. Iran.

Email: D_MTehrani@sbu.ac.ir

Tel: +98-21-29903144

Fax: +98-21-22431664

Number of words: 2736

Number of figures: 5

Number of tables: 0

Number of references: 19

1

Abstract

Tramadol is an analgesic drug that binds to specific

opioid receptors. It may contribute to the inhibition of

neuronal re-uptake of noradrenaline. Catalase is a key

enzyme for degrading H2O2 in cells and has various

isoforms with different structures and kinetics

properties. In this research, the effect of tramadol on

the activity of catalase of Pseudomonas and mouse liver

was investigated and compared. Tramadol could inhibit

Pseudomonas catalase with mixed inhibition while

inhibiting mouse liver catalase in a non-competitive

manner. The Ki and IC50 values were determined as 0.45 and

1.5 mM for Pseudomonas and 2.8 and 2.5 mM for mouse liver,

respectively. SDS-PAGE of partially purified catalases

determined different molecular weights for Pseudomonas (Mw

54 kDa) and mouse liver (61 kDa).

Keywords: Drug, Enzyme, Inhibition, Mouse liver,

Bacteria.

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Introduction

Tramadol is a centrally acting synthetic opioid analgesic

which possesses opioid agonist properties and activates

monoaminergic spinal inhibition of pain. It may be

administered orally, rectally, intravenously or

intramuscularly [1, 2]. The drug may become a useful

alternative for the opioid analgesics which are currently

available for the treatment of patients with moderately

severe acute or chronic pain [1].

Experimental and clinical data have suggested that

tramadol may also exert its analgesic effect through

direct modulation of central monaminergic pathways [2].

Clinical experience has confirmed that tramadol is an

effective and relatively safe analgesic that may be of

value in several pain conditions, not requiring treatment

with strong opioids [2]. Tramadol is a synthetic analogue

of codeine that binds to mu opiate receptors and inhibits

norepinephrine and serotonin reuptake [3].

Tramadol has dose- and time-dependent bactericidal

activity against E. coli and S. epidermidis as well as

antibacterial activity against S. aureus and P. aeruginosa [4-

3

6]. It also possesses a significant antibacterial

activity against E. coli in urinary tract infections [6].

Catalase (CAT) (EC 1.11.1.6) is a heme containing enzyme

that catalyzes decomposition of hydrogen peroxide to

water and oxygen and is found in almost all cells, except

certain anaerobic bacteria. Bovine CAT was one of the

first enzymes to be isolated up to a high state of purity

and the first iron-containing enzyme which was isolated

[7]. CAT has many varieties in different organisms. Most

of catalases are monofunctional which only decompose H2O2.

Others are bifunctional catalase-peroxidases and

manganese-containing catalases [8].

H2O2 is a potent oxidative agent and is normally produced

during cell metabolism. H2O2 can damage the cells through

oxidation of nucleic acids, unsaturated lipids and

proteins [9]. CAT plays a main role for removing H2O2 and

preventing cell injures. The CAT activity can be affected

by some compounds; consequently, the cells may be injured

due to change of H2O2 concentration.

It has been shown that some chemicals and drugs inhibit

CAT activity. Some heavy metals such as copper inhibit

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CAT with a non-competitive pattern [10] and cyanide acts

as a competitive inhibitor [11, 12]. The inhibition of

CAT by nitric oxide and relationship of this effect with

nitric oxide cytotoxicity were also investigated [13].

Some drugs have been also found to have an inhibitory

effect on CAT activity. Human skin CAT activity was

inhibited by vesnarinone, piroxicam, ketoprofen,

diclofenac sodium and nidazole [14, 15].

Although there are some reports on the CAT inhibition by

drug, there is no work on the kinetic aspects of drugs'

action on CAT.

In this report, for the first time, tramadol was

introduced as an inhibitor for CAT and also the kind of

inhibition and kinetics parameters of interaction was

determined. Besides, a comparison of the interaction of

tramadol between CAT in Pseudomonas aeruginosa and mouse

liver was investigated, which opened a new view to

understanding the behavioral difference between isoforms

of the enzyme.

Materials and Methods

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All the chemicals used for buffers' preparation, enzyme

assay and electrophoresis were purchased from Merck

Company. DEAE cellulose and tramadol hydrochloride were

provided from Sigma Company. Sephadex G-100 was obtained

from Pharmacia Company.

Preparing cell free extract

Balb/C mouse liver was sliced to small pieces and washed

with NaCl (9 g/L) solution. The mouse liver pieces were

homogenized and centrifuged (for 10 min at 3000 × g) to

remove intact cells. The supernatant was used as the cell

free extract for enzyme assay.

Pseudomonas aeruginosa (ATCC 31479) was cultured in the

minimal medium containing 2.5 g KH2PO4, 2.5 g Na2HPO4, 1 g

NH4NO3, 0.2 g MgSO4 and 0.01 g CaCO3 per liter adjusted to

pH 7.0. Ethanol was added to this medium to the final

concentration of 1% (v/v) as a carbon source. The

bacteria were cultured for 72 h in a reciprocal shaker at

150 rpm at 30°C. After 72 h (in the stationary phase),

the medium was centrifuged (5000 × g for 30 min) to

precipitate cells. The precipitated cells were washed

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twice with phosphate buffer 0.1 M, pH = 7 and centrifuged

(7000 × g, 30 min at 25°C). The pellet was dissolved in

the phosphate buffer to obtain cell suspension. To break

the cells, bacteria suspension was sonicated for 5 × 20

sec with 20 sec intervals at 4°C. The sonicated

suspension was centrifuged (12000 × g for 30 min at 0-

4°C) to precipitate intact cells. The supernatant was

used as the cell-free extract for further experiments.

Enzyme assay

H2O2 was used as substrate to measure CAT activity.

Tramadol was prepared with concentrations ranging from

0.65 to 3.8 mM as an inhibitor. The reaction was started

by adding 100 μl of the cell-free extract which contained

CAT to test tubes containing 0.1 M phosphate buffer (pH =

7) and different concentrations of H2O2 as substrate (1 -

8 mM) for 10 min in the presence and absence of tramadol.

The final volume in each test tube was always 2.2 ml. The

catalytic activity of CAT was monitored continuously by

following the decrease in absorption at 240 nm using a

UV-visible spectrophotometer (Shimadzu 1240). CAT

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activity was measured using the extinction coefficient of

43.6 M−1 cm−1 for H2O2 at 240 nm [16]. Lineweaver-Burk plot

was used to obtain Km and Vmax of the enzyme. The reaction

rate of the enzyme was also examined at different pHs (3,

4, 5, 6, 7, 8, 9, 10, 11 and 12). Different buffer

systems including glycine, phosphate, acetate and Tris

were used to obtain 0.1 M buffer in which pH varied from

3 to 12.

Partial purification of enzyme

The cell-free extract from mouse liver and Pseudomonas

were brought to 50% ammonium sulfate saturation. The

precipitate was collected by being centrifuged at 5000

g for 10 min and dissolved in 0.1 M phosphate buffer (pH

= 7). The suspension was dialyzed against phosphate

buffer overnight. All the above procedures were performed

at 4°C. The dialyzed suspension was loaded onto a DEAE

cellulose column equilibrated by 50 mM Tris buffer (pH =

8). Elution was performed by increasing NaCl

concentration at the flow rate of 1 ml/min. The fractions

were monitored for the amount of protein (at 280 nm

8

absorption) and enzyme activity. The fraction with

maximum activity was selected for gel filtration

chromatography which was performed by Sephadex G-100. The

fractions with maximum activity were selected for SDS-

PAGE electrophoresis.

Results

Inhibition pattern

The reaction rate of CAT was determined in the absence

and presence of tramadol by monitoring the reduction of

substrate (H2O2) at 240 nm. The incubation of enzyme with

H2O2 continued to reach the plateau (substrate depletion).

In the presence of tramadol, the reaction rate of enzyme

in both mouse and bacterium was reduced which determined

that the drug inhibited the enzyme activity. Lineweaver-

Burk plot was applied to find out the inhibition pattern.

The inhibition in bacterium was different from that in

mouse CAT. Tramadol inhibited Pseudomonas CAT with mixed

pattern (Fig. 1 left) while it inhibited mouse liver CAT

with constant Km (non-competitive) (Fig. 1 right).

The Km of enzyme was increased by increasing tramadol

concentration and IC50 value was determined to be 1.6 mM

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in Pseudomanas (Fig. 2 left). In mouse liver, Vmax of the

enzyme decreased by increasing the drug concentration and

the IC50 value was about 2.4 mM (Fig. 2 right). The slope

of lines and the intercepts from Lineweaver–Burk plot

were used to determine the Ki of the inhibitor in

Pseudomonas (0.45 mM) and mouse liver (2.8 mM),

respectively (Fig. 3).

pH profile

The effect of pH on CAT activity was compared in

Pseudomonas and mouse liver (Fig. 4). Both CATs had the

maximum activity at pH 7. In mouse liver, another peak

was observed at pH of 9 and the activity was suddenly

decreased at pH of 10. In Pseudomonas, there was another

peak at pH=10 and the enzyme remained active at pH=11;

but, no activity was observed at pH=12.

SDS-PAGE

SDS-PAGE was performed to compare the mobility and

molecular weight difference between the two CATs. The

molecular weight of bacterium CAT was determined to be

10

about 54 kDa while the CAT of mouse liver was calculated

to be 61 kDa (Fig. 5).

The specific activity of partial purified enzyme was

determined to be 9.65 and 28.5 U/mg protein in Pseudomonas

and mouse liver respectively.

Discussion

Tramadol is a narcotic-like pain reliever drug which is

widely used to treat moderate to severe pains. One of the

most important precautions for drug consumption is drug

side effect that can affect body physiology. Most of the

drugs may interact with receptors or enzymes other than

the targets for which they have been designed [17, 18].

In this regard, it was shown that cimetedine inhibited

renal alkaline phosphatase with un-competitive manner

[17]. This report focused on the effect of tramadol on

the activity of CAT. In this research, the inhibitory

effect of tramadol on CAT activity was reported for the

first time and the kinetics aspects of interaction were

determined in Pseudomonas and mouse liver. Mouse liver was

chosen as a model for eukaryotic CAT and was compared

with Pseudomonas as a model for prokaryotic cells.

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Although the effect of some drugs such as piroxicam,

ketoprofen, diclofenac sodium and nidazole on human skin

CAT has been reported [15], no reports has indicated the

kinetics aspect of inhibition. It was shown that CAT had

various isozyme in animals, plants and microorganisms,

which had various structure and properties [8, 19].

The present results showed that tramadol could inhibit

CAT activity and the pattern of inhibition was different

in Pseudomonas and mouse liver. Tramadol inhibited

Pseudomonas CAT with mixed pattern while it inhibited

mouse liver CAT with non-competitive manner. These

results suggested that tramadol could bind to the

bacterium and mouse liver CAT from the site other than

active site. The comparison of Ki and IC50 values of

tramadol in both of the samples showed that the drug

could bind to liver CAT with higher affinity than the

bacterium CAT. Not only the two CATs showed dissimilar

behavior in kinetics parameters but also they

demonstrated a different pattern in their pH profiles. In

high basic pH, Pseudomonas CAT was more resistant than the

mouse liver CAT. The former was active at pH=10 and 11

12

while the latter lost its activity at pH=10. This

suggested that the two CATs might have different

structures and be considered as two isoforms.

Consequently, the drug could inhibit them with different

manners. To confirm the structural difference between

these two CATs, SDS-PAGE from partial purified enzyme was

performed. The results showed that the two CATs did not

have the same mobility and had different molecular

weights. This suggested that the two enzymes had

different structures. The molecular weight of CATs

obtained from SDS-PAGE was very similar to Pseudomonas

(55kDa) and bovine liver (60kDa) molecular weight CTAs

which was reported by Switala and Loewen [19].

That report also indicated the difference kinetics

parameter of binding of some CAT inhibitors such as KCN,

NaN3 and Aminotriazole between various CATs [19].

In conclusion, this was the first report that compared

the inhibition pattern of the CAT isoform in eukaryotic

and prokaryotic cells. Tramadol was introduced as an

inhibitor of CAT and its kinetics parameters were

determined. The CAT of Pseudomonas was partially purified

13

and its molecular weight was resolved and compared with

that of mouse liver CAT.

14

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Legends:

Figure 1: Lineweaver-Burk plots were obtained for

determining the pattern of inhibition. Left: Inhibition

in Pseudomonas CAT (mixed). Right: Inhibition of mouse

liver CAT (non-competitive).

Figure 2: Determination of IC50 values in Pseudomonas CAT

(left) and mouse liver CAT (right).

Figure 3: Secondary plot derived from Lineweaver-Burk was

used for Ki determination. For Pseudomonas (mixed

inhibition) the slopes of lines (left graph) and for

mouse liver (non-compatitive) the ordinate intercept of

lines (right graph) were utilized to calculate the Ki of

tramadol.

Figure 4: Comparison of pH curves in Pseudomonas CAT and

mouse liver CAT.

Figure 5: Silver nitrate stained SDS-PAGE of partial

purified CAT of Pseudomonas and mouse liver. Lane1:

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Protein standard, Lane 2: CAT of Pseudomonas (P), Lane 3:

CAT of mouse liver (M), Lane 4: Bovine serum albumin with

Mw 66500.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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