The influence of composition and structure of water-in-oil microemulsions on activities of Iraqi Turnip peroxidase

2.1 Materials

Peroxidase (specific activity 5333 units/mg protein) was produced from germinated seeds of Iraqi Turnip by Sakran (2008). Purified water was taken from a Millipore Milli-Q system (electrical conductivity < 10⁻⁶ S m⁻¹). The buffer contained phosphate solution (concentration c = 0.05 mol/l), pH 7.4. Chloroform and series alcohols used from 1-propanol to 1-hexanol (purity > 99%) were purchased from Merck. SDS (purity > 99%) was obtained from El-nasr pharmaceutical chemicals Co. The substrates chosen were hydrogen peroxide (was purchased from Merck, 30% (w/w) medical. Extra pure) and guaiacol (was supplied from sigma chemical company, USA).

2.2 Preparation of microemulsion

Microemulsions were prepared by the addition the desired amount of surfactant mixture in which SDS and alcohols of the homologous series 1-propanol to 1-hexanol were mixed at a weight ratio of 2:1 to chloroform (10 ml). In this system, the phosphate buffer solutions (0.05 mol/l, pH 7.4) were then added to, and the final ω₀ value was adjusted by the addition of the amount of the buffer. The mixture was briefly shaken until an optically clear single-phase solution was formed.

2.3 Preparation of the reaction mixtures

The reaction mixtures were prepared by the addition 10 μl of a solution containing desired concentration of guaiacol to 1 ml of the microemulsion media. Then 0.5 ml of a solution containing 9 mM H₂O₂ and finally 50 μl of a solution containing ITP (330 μg/ml) were added. The final addition of ITP initiates the oxidation of the guaiacol. The concentrations of ITP and guaiacol in the microemulsion reaction system were 16.5 μg/ml and ranging from 0.08 to 0.86 mM, respectively.

2.4 Determination of enzyme activity

The kinetics of guaiacol oxidation was studied spectroscopically at a constant temperature of 25 °C using a varian cary 3E spectrometer. The temperature (T) of cell compartment was maintained with Haake circulator KT 33. The progress of the reaction was estimated from the absorption at 470 nm of oxidized form of guaiacol, which was detected during the first 2 min after the addition of ITP. One enzyme unit is the change in molarity (M) per minute under assay conditions. Molar extinction coefficient, 26,600 M⁻¹ cm⁻¹ was used to convert absorbance into molarity (Keyhani et al., 2000).

3.1 Effect of “water pool” size in w/o microemulsion and enzyme concentration on enzyme activity

The size of water pools in w/o microemulsion occurs to be the most important factor that determines the reaction rate and it has been studied recently (Jing et al., 2003). The size of water pools in w/o microemulsion strongly dependent on ω₀. Altering ω₀ influences on stability of the microemulsion. At a constant overall enzyme concentration ([E]_ov) of 16.5 μg/ml and constant total SDS concentration of 0.014 mol/l, the effect of ω₀ on the ITP activity in the w/o microemulsion was determined under the conditions of pH 7.4 and 25 °C. The experimental results showed bell-shaped curve with a maximum value of V at ω₀ = 8 as presented in Fig. 1. In the system under study, the peroxidase contacts with the water pool interface and guaiacol is partitioned between the organic phase and interface. Therefore, the reaction most likely carried out at interface or close to the interface and the peroxidase concentration in water pool may affect the initial reaction rate. With [E]_ov, SDS concentration, substrate concentration, pH, and temperature being fixed at 16.5 μg/ml, 0.014 mol/l, 0.03 mol/l, 7.4 and 25 °C respectively; the initial reaction rates were measured for various concentrations of ITP in water pool ([E]_wp) and are presented in Fig. 2. The experimental results illustrated that the activity of ITP in the SDS microemulsion system had a typical bell-shaped dependence on [E]_wp with a maximum value of initial reaction rate at [E]_wp = 16.2 μg/ml. The existence of optimal ω_o for ITP activity was probably related to conformational changes of the protein, to the changes in the water structure in the hydration shell, and to the water activity in the microemulsion (Jing et al., 2003).

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Figure 1

Dependence of V/[E]_ov on ω₀ at 25 °C, pH = 7.4, [guaiacol] = 0.03 mol/l, [SDS] = 0.014 mol/l, and [E]_ov = 16.5 μg/ml in SDS/1-hexanol/CHCl₃/buffer system.

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Figure 2

Dependence of initial reaction rate on enzyme concentration in the water pool at 25 °C, pH = 7.4, [guaiacol] = 0.03 mol/l, [SDS] = 0.014 mol/l, and [E]_ov = 16.5 μg/ml in SDS/1-hexanol/CHCl₃/buffer system.

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3.2 Effect of the number of carbon atoms in the alcohols on enzyme activity

The influence of the alkyl chain length of 1-alcohols on the enzymatic activity was tested in w/o microemulsion. This was done to evaluate the maximal enzymatic inhibitions caused by each alcohol. As shown in Fig. 3, the results exhibited the maximal enzymatic activity in presence of 1-hexanol. Thereby, an increase of inhibition was observed for shorter alcohols (1-propanol and 1-butanol). The denaturation of horse radish peroxidase by 1-alcohols from 1-butanol to 1-octanol has been extensively studied (Bauduin et al., 2005). It was concluded that the inhibition influence of the alcohols depends both on the solubility of the alcohols in the buffer and the flexibility of the interfacial film which is usually expressed in terms of an elastic constant or an elastic modulus (De Gennes and Taupin, 1982). The very low enzymatic activity values in the SDS/1-propanol/CHCl₃/buffer system can be explained by the relatively high solubility of 1-propanol in the aqueous buffer system (solubility of 1-propanol in water at 25 °C (w/w) = 0.0740). Due to 1-propanol is essentially dissolved in the aqueous droplets leading to a strong inhibition of the water soluble enzyme. Moreover, 1-propanol acts as cosurfactant with the particular feature that the SDS/1-propanol film is a highly flexible. It is also possible that ITP has a certain affinity to the interface. As consequence, the affinity of the enzyme to the highly flexible film also exposes the enzyme to the denaturating chloroform phase, a fact which further decreases the activity.

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Figure 3

Dependence of initial reaction rate on n, the number of carbon atom of the 1-alcohol as co-surfactant.

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In SDS/1-hexanol/CHCl₃/buffer system, the initial enzymatic activity increases up to a maximum. The high V value of 12.78 μM min⁻¹ in the 1-hexanol system is due to the low solubility of 1-hexanol in water (solubility of 1-hexanol in water at 25 °C (w/w) = 0.0058). It is known that 1-hexanol yields a more rigid film than shorter alcohols do (Bauduin et al., 2005) and obviously the 1-hexanol molecules are so strongly incorporated in the film that their inhibition effect is negligible.

3.3 Effects of substrate concentration on enzyme activity

The enzyme-catalyzed kinetics has been investigated in SDS-based w/o microemulsions (SDS/1-hexanol/CHCl₃/buffer). It was well known that enzyme-catalyzed reactions usually obey Michaelis–Menten kinetics represented by the following equation:

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Figure 4

Dependence of initial reaction rate on substrate concentration at 25 °C, pH = 7.4, ω₀ = 8, and [E]_ov = 16.5 μg/ml in SDS/1-hexanol/CHCl₃/buffer system.

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Figure 5

Lineweaver–Burk plot of Iraqi Turnip peroxidase action on guaiacol in the presence of hydrogen peroxide at 25 °C, pH = 7.4, ω₀ = 8, and [E]_ov = 16.5 μg/ml in SDS/1-hexanol/CHCl₃/buffer system.

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