Introduction
Phosphatases are enzymes that catalyze the hydrolysis of orthophosphate monoesters with consequent release of inorganic phosphate and are astray distributed in nature.
The seeds of plants are a particularly rich root word of typical acid phosphatases. The amount of phosphatase activity in seeds normally increases sharply upon germination and then falls as the seedling develops. The hairsplitting physiological function of this phosphatase is not known, but it is probably active in releasing phosphate from organically bound storeho handling forms, such as inositol hexaphosphate (phytic acid), for use in the metabolism of the germinating seedling.
In this experiment, acid phosphatase from wheat germ is apply to develop skills in assaying and studying general kinetic properties of enzymes. The assay to be used takes advantage of the broad specificity of the phosphatase by using the artificial substrate, p-nitro-phenyl phosphate (pNPP). The degree of hydrolysis of the substrate is determined by photometric amount of the p-nitrophenol liberated in the reaction. In alkaline solution the p-nitrophenolate ion absorbs cast down strongly in the region of 405 nm.
Since you are able to use a spectrophotometer and standard cells with a 1.0 cm light cut , you can use the extinction coefficient for p-nitrophnol in 0.02M NaOH ( ? = 18.
8x103 M-1cm-1 at 405 nm) to describe the (moles of product formed from your absorbance readings.
Materials and Methods
A. Materials:
1. 0.06 M Disodium p-nitrophenyl phosphate (pH 4.8), (pNPP, keep cold)
2. 0.5 M Sodium hydroxide
3. 0.05 U/ml Acid phosphatase in 0.1% bovid serum albumin.
4. 0.025 M Sodium phosphate (pH 4.8)
5. 0.05 M citrate buffer (pH 4.8)
B. Equipment:
1. Test pipings
2. Pipette and tips
3. Water bath
4. Test tube rack
5. Spectrophotometer
C. Procedure:
A. Determination of the Michaelis Constant:...If you want to get a full essay, order it on our website: Ordercustompaper.com
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