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Sunday, December 13, 2009

How to Estimate Phosphataes

Phosphataes are enzymes capable of cattily zing the hydrolysis of certain organic phosphates. The general reaction may be written as.




Depending upon PH optima, they are classified on acid or alkaline phosphates. An acid phosphate has PH optima between 7.6 and 9 depending upon their origin. The principle of measurement of activity is the determination of product of hydrolysis of the substrate liberated under carefully standardized conditions. Hence, the hydrolytic activity can be determined by estimating the inorganic phosphate. Inorganic phosphate reacts with (NH4) MoO4 in an acid solution to form phaspho molly bdic acid. A reducing agent such as Fiske-Subbarow reagent is added to reduce the Mo+ r to Mo+ 4 phosphomolly bdous acid which gives a characteristic blue co lour but does not affect the uncombined moly bdic acid.


Phosphates activity is determined by estimating the amt. of product of enzymic activity formed in a certain time under defined conditions of temperature, PH and salt content. One of the most widely used units of phosphates activity is defined as amount of enzyme which liberate 1 gums of phosphate. Phosphorous as phosphate ion during the 1st hour of incubation as 370C and at PH 5.4 from the substrate [No – B – glycerophosphate].

Requirement:-
1) Sodium – B – glycerophosphate
2) Buffer at PH 5.4.
3) 10 N H2SO4 .
4) Fisk subbarows reagent.
5) 10% TCA
6) 2.5% (NH4)2 MoO4
7) Standard phosphate solution [KH2PO4 solution] 80 gms / ms.
8) Enzyme source.


Procedure: - Prepare a series of test tubes containing different amount of monk-phosphate [KH2PO4] oil stings in range of 0 – 40 llg / ml of phosphorus. Make up the volume to 3 ml with distilled water. Add 0.4 ml of 10 NH2SO4 0.8 ML OF 2.5% NH4 – meliorate and 0.4 ml of Fisloe – subbarow’s reagent. Dilute the solution to 10 ml and read extinction of 640 nm after 10 – 15 mins.
Sample: - Prepare 5% homogenate of germinated moong and centrifuge use supernatant as enzyme extract.
Prepare reaction mixture as follows:
Blank[S]: Buffer 1 ml + 0.2 ml enzyme extract + 0.8 ml water.
Sample: Buffer 1 ml + 0.2 ml enzyme extract + 0.2 ml subtask + 0.6 ml water.


Incubate the tubes at 370 C for 1 hour. 0.5 ml tichboro acetic acid is added to inactivate the enzyme, use this solution as the sample solution of phosphate and determine the absorbance.
136.1 llgm of KH2PO4 == 98 gms of H3PO4 = 30.975 ugmsot – p.



The activity of acid phosphates is found to be 128 units 1 ml at optimum PH and temperature.

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