ENCAPSULATION OF CATALASE AND PEG-CATALASE IN ERYTHROCYTE

Abstract

Reactive partially reduced oxygen species such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH) are produced in aerobically growing organisms during normal cellular respiration. To provide an effective defense against these reactive species, many aerobic organisms have evolved a multienzyme defense which includes superoxide dismutase, catalase and peroxidase. The superoxide anion may cause appreciable cellular damage by oxidizing aminoacids or by causing DNA strand breakage. Catalase was covalently immobilized on activated methoxypolyethyleneglycol-5000 and catalase and PEG-catalase were encapsulated in erythrocyte. Enzyme activity, encapsulation yield and hemograme analysis were determined for each sample. The erythrocyte shape of the samples were investigated by using phase contrast microscopy.

INTRODUCTION

Catalase that is usually found as a tetrameric feriheme enzyme (EC 1.11.1.6) is one of the most extensively studied enzyme. It is widely distributed in nature and is found in all microorganisms as well as plants and animal cells [1-2]. Catalase can be found in human tissues with its highest activity in the erythrocytes, liver and kidney, while its activity is very low in human serum. In tissues catalase is localized in the mitochondria and peroxisomes, while erythrocytes it can be found in soluble form. Its physiological function is not quite clear, but its protective role against free radicals from oxygen has been emphasized.

Aerobic metabolism entails the production of reactive oxygen species, even under basal condition, hence there is a continous requirement for inactivation of these reactive oxygen species. The steady-state of prooxidants and antioxidants may be disrupted. A disbalance in favor of the prooxidants and disfavoring the antioxidants, potentially leading to damage, has been called “oxidative stress” [3-4] such damage may afflict all types of biological molecules, including DNA, lipids, proteins and cabohydrates. Thus oxidative stress may be involved in processes such as mutagenesis, carcinogenesis, membrane damage, lipid peroxidation, protein oxidation and fragmentation, as well as carbohydrate damage [[5]].

All cell in eucaryotic organism contain powerful antioxidant enzymes. The three major antioxidant enzymes are superoxide dismutase, catalase and peroxidases. In addition, there are numerous specialized antioxidant enzymes reacting with and in general, detoxifying oxidant compounds. In direct antioxidant functions carried by enzymes are (a) the back up function and (b) the transport and elimination of reactive compounds [[6]].

Erythrocytes posses many desirable properties of a suitable carrier. They are biodegradable and circulate throught circulatory system; large quantities of biologically active material can be encapsulated in relatively small volumes of these cells; they can be organ targeted within the reticuloendothelial system [7-9]and they can be fused with living cells [[10]]. Catalase and PEG-catalase were encapsulated in erythrocyte (1/1) (v/v) by using slow dialysis methods and some parameters were investigated.

MATERIALS AND METHODS

Chemicals

Methoxypolyethyleneglycol-5000, catalase were purchased from Sigma chemical; H2O2 was obtained Merck. All other chemicals and organic solvents were obtained from commonly used suppliess and were analytical grade or better.

Preparation of PEG-Catalase

MPEG-5000 was activated with cyanuric chloride and coupled to catalase by our previously study [12-13]. The extent of conjugation was determined by measuring the number of reactive amino groups in the solution by TNBS methods [[13]].

Enzyme Assay

Due to the absorption of H₂O₂ at 240 nm, the activity of free and PEG- catalase and encapsulated enzymes have been measured spectrophotometrically [[16]]. We used 10.5 mM H2O2 solution as substrate which is prepared in phosphate buffer pH 7.4 50 mM. For the activity measurement of enzyme the reaction was begun by adding 0.02 ml of enzyme to the 3 ml of substrate in the cells. The absorbans values has determined each 10 seconds and the activity values were estimated by using first absorbance and eq (1);

Physical Characterization of Catalase and PEG-Catalase

Effect of pH and temperature on the activity of free and immobilized catalase were determined.

Preparation of Red Blood Cells

Human erythrocytes were isolated from freshly drawn, heparanized verous blood. Whole blood was centrifuged at 2000xg for 10 min. The serum and buffy- coat were removed and the packed cells were washed three times in 10 vol of phosphate buffer saline (10 mM pH 7.4, 0.15M NaCl, 10 mM glucose, 10 mM inosine) [[13]] to remove unentrapped material.

Encapsulated Catalase and PEG-Catalase

Catalase and PEG-catalase encapsulated erythrocytes using slow dialysis procedure as described by Kruse et al. with optimization by our previously study [[11]], [[13]]. Percentage entrapment, percentage equlibration, cell yield, mean cell volume (McV), mean cell hemoglobin (McH), hematocrit (HcT), mean corpucular hemoglobin (McHc) were determined for each samples on a hematological analyser Celldyn 4000. The erythrocyte shape of the encapsulated samples were investigated by using phase contrast microscopy.

RESULT AND DISCUSSION

Catalase Immobilization on Activated PEG-5000

Catalase was covalently immobilized on activated PEG-5000. after immobilization total activities of each immobilized catalases were shown in table 1.

Table I. Immobilization of Catalase on Activated PEG

Molar ratio (catalase-Lys/activated PEG)	Remaining Act. (%)	∼Blocked amino groups (%)	∼Molecular weight (DA)
1/1	71.0	23.0	368.000
1/3	59.0	36.0	440.000
1/7	47.0	53.0	532.000

Physical Characterization

The effect of pH on the activity of immobilized catalase (1/3) and free catalase were studied in the different buffer solution of pH varying 4–10 (fig. 1). The optimum pH was found to be the same pH 7.0 for both enzymes. Optimum temperature of immobilized and free catalase were found to be 37°C (fig. 2).

Figure 1. Optimum pH.

Figure 2. Optimum temperature.

Encapsulation of Catalase and PEG-Catalase

Catalase and PEG-catalase encapsulated within erythrocyte using different molar ratio. Encapsulation yield and the amount of encapsulated catalase were investigated. As is shown in table 2 and 3 max. entrapment amount of catalase was given by using 2 mg/ml catalase concentration and percentage entrapment and the amount of encapsulated catalase are increased by using PEG as PEG is a hydrophobic molecule. Also, PEG protect the shape of erythrocyte but if catalase concentration was increased erythrocyte shape has been defect (fig. 3f)

Table II. Encapsulation of Catalase in Erythrocyte

Catalase conc. (mg/ml)	Catalase activity (U)	Catalase activity in sample (U)	Encapsulation yield (%)	The amount of encapsulated catalase (mg)
1.0	542.21	162.33	29.29	0.29
2.0	2925.75	366.40	12.52	0.63
3.0	1662.64	157.78	9.49	0.45
5.0	2771.06	234.71	8.47	0.25

Table III. Encapsulation of PEG-catalase in Erythrocyte

PEG-Catalase conc. (mg/ml)	PEG-Catalase activity (U)	PEG-Catalase activity in sample (U)	Encapsulation yield (%)	The amount of encapsulated catalase (mg)
1.0	506.73	415.18	79.99	1.20
1.9	1266.83	1135.80	86.66	4.25

Catalase activity in sample = amount of encapsulated catalase act. − amount of catalase act. in control erythrocyte.

Figure 3. The shape of erythrocyte samples (3a-1mg/ml catalase, 3b-1mg/ml PEGcatalase, 3c-2mg/ml catalase, 3d-1.9mg/ml PEGcatalase, 3e-5mg/ml catalase; magnification 3.3 × 10).

Several parameters of the erythrocytes submited to the procedure described above were investigated before and after encapsulation procedure. The hematologic parameters of encapsulated and control RBC from a typical preparation are presented in table 4 for catalase and table 5 for PEG-catalase. As is shown in table 4 and 5, there was a larger drop in McV and the decrease in McV for encapsulated mouse, bovine and human erythrocytes [[19]], [[13]]. With the reduction in the McV, there was an associated decrease in hemoglobin concentration. It appears that the lost of HgB and other cellular contents during the lysis step may account for the drop of the McV value of the encapsulated erythrocyte. We obtained max cell recovery using 2 mg/ml catalase and PEG-catalase and this recovery is very good. As is shown in table 4 and 5, using of PEG in enzyme encapsulation allowed to enter the catalase with the high loading capacity into the erythrocyte [[13]].

Table IV. Hematologic Parameters of Encapsulated Catalase and Control RBC

Parameters	Control	1mg/ml catalase	2mg/ml catalase	3mg/ml catalase	5mg/ml catalase
RBC	0.51	0.51	0.50	0.48	0.40
HgB	1.30	1.05	0.97	0.89	0.80
HcT	3.90	2.77	2.34	2.53	2.77
McV	77.5	63.1	55.0	53.9	50.6
McH	25.2	19.2	20.8	18.6	19.8
McHc	33.3	37.9	41.4	35.3	28.1
Cell recovery (%)		99.4	98.0	94.1	78.2
Hemoglobin release(%)		19.3	25.3	31.5	38.4

0.1 ml sample + 1.9 ml isoton.

Table V. Hematologic Parameters of Encapsulated PEG-Catalase and Control RBC

Parameters	Control	1 mg/ml catalase catalase	1.9 mg/ml catalase
RBC	0.51	0.51	0.45
HgB	1.30	1.15	0.94
HcT	3.90	3.61	2.66
McV	77.5	73.9	59.7
McH	25.2	29.5	16.5
McHc	33.3	39.9	27.7
Cell recovery (%)		99.0	87.5
Hemoglobin release (%)		11.5	28.0

0.1 ml sample + 1.9 ml isoton.