Acid phosphatase refers to a group of enzymes which catalyze the hydrolysis of certain monophosphoric esters, a reaction of considerable importance in several body processes, have been isolated from animals and plant tissues as well as from bacterial and fungal cells (Besford, 1979; Herman and Liss, 1963; Fatima et al., 1981; Srivastava and Gupta, 1986). Acid phosphatases exhibit optimum activity at pH 4.2-7.4.
Higher acid phosphatase in different wheat varieties are recognized as cold resistance. Acid phosphatase activity is also used in plant breeding as an indicator of cold resistance (Hagima, 1984). So far as we know the detailed kinetics of the enzyme for plant tissues have not been studied as yet, so a study of acid phosphatase isolation and characterization was undertaken.
Materials and Methods
Preparation of the Enzyme: The grains of wheat (Pirsabak-85) were cleaned and ground to a fine powder. The crude enzyme was extracted by shaking powder in 0.1 M acetate buffer (1 g/10 ml) of known pH for 1 h at 30°C. The mixture was then centrifuged at 5000 rpm for 5 min. The supernatant thus obtained was used for further work.
Estimation of Enzyme Activity: Acid phosphatase activity was determined by method of Gutman and Gutman (1940) using P-nitrophenyl phosphate as substrate. P-nitrophenyl phosphate, as the result of hydrolysis was converted into phenolate ions (Yellow Colour) at alkaline pH. The yellow colour was read at 405 nm. 900 μl of ffie substrate (0.1 M) was incubated for 5 min at 30°C. Then 100 μl of enzyme was added to it and mixed. The mixture was further incubated for exactly 5 minutes. The reaction was stopped by adding 4 ml of sodium hydroxide (0.1 M). The yellow product of P-nitrophenolate anions formed was read at 405 nm against the blank, which was prepared by adding 4 ml sodium hydroxide and 100 μl buffer to incubated substrate (900 μl). To increase, precision we recorded the transmittance and then converted it to absorbance according to the formula:
A = 2-Log % T
The activity was expressed as milli mole of substrate hydrolyzed per minutes.
Results and Discussion
Effect of pH on the Activity: Acid phosphatase was extracted from wheat grains at different pH values. The results are given in Fig. 1. which suggest that maximum extraction of the enzyme is achieved at pH 5.5. However, for maize the maximum activity of the enzyme was released at pH 8.5 (Fatima et al., 1981). Since these workers used a different extraction medium of different ionic strength, the release of the enzyme seems to depend on the nature as well as on concentration of the medium or on its binding in the tissue. Consequently, a pH 5.5 was used thought, both for extraction as well as for estimating enzyme activity.
|Fig. 1:||The effect of pH on activity
Effect of temperature on Enzyme Activity: The enzyme activity was determined at different temperatures varying from 20-70°C. Fig. 2 shows that the enzyme has maximum activity at 30°C. Above this temperature, the activity was decreased sharply which may be due to heat denaturation of the enzyme.
Effect of incubation time on the enzyme activity: Acid phosphatase activity was determined after different incubation times. The results are shown in Fig. 3 which indicate that activity was linear function of time during at least 10 min. After this, activity increased but not proportionately. The deviation may be due to product inhibition.
|Fig. 2:||The effect of temperature on activity|
|Fig. 3:||The effect of incubation time on activity|
Effect of substrate concentration on the enzyme activity: The effect of substrate concentration on enzyme activity was studied with P-nitropenyl phosphate as substrate using concentration between 10 pM and 400 pM. With increase in concentration of the substrate the enzyme activity increased for upto 200 pM. Further increase of substrarte concentration slightly decreased the enzyme activity, showing that enzyme was inhibited. The Michaelis-Menten constant lq (Webb 1963; Mabood et al., 1984) was determined by Hanes plot as showing in Fig. 4, the value was found to be 10 pM.
|Fig. 4:||The effect of substrate concentration based on S/V versus S plot|
Effect of enzyme concentration on activity: The effect of enzyme concentration on activity was assayed. The results are shown in Fig. 5. It is evident that the enzyme activity was linear for upto 85 percent of the original enzyme concentration (extract). Hence in further work 85 percent of the enzyme concentration was used for assaying enzyme activity.
|Fig. 5:||Effect of enzyme concentration on activity|
Effect of in vitro storage: To investigate the effect of the in vitro storage on the activity of the enzyme, the extract of pH 5.5 was stored at 40°C over a period of one week. The results are given in Table 1. It is noticed that there is no change in activity of the enzyme for upto the period of storage. This sort of ability of the enzyme suggests that it is a suitable enzyme for advanced kinetic. It is concluded that wheat is a good source of acid phosphatase and that the enzyme may be suitable one for advanced kinetic studies. Further purification and characterization as well as detailed kinetic studies of the enzyme are in progress.
|Table 1:||The effect of storage on activity|