Isolation and Characterisation of Maize Acid Phosphatase

Topics: Enzyme, PH, Enzyme assay Pages: 14 (3830 words) Published: September 8, 2013
Isolation and Characterization of Maize Acid Phosphatase|
BCHM 405|
Aaron BANESEH 10307275|

Table of Contents
1Table of Contents2
2Introduction4
3Experimental Procedures5
3.1Sample preparation5
3.2Initial Extraction of Acid Phosphatase5
3.3Determination of Protein Concentration by the Folin-Lowry Assay5
3.4Specific Activity Assay6
3.4.1Definition of Enzyme Unit and Specific Activity-6
3.5Determination of pH Optimum6
3.6Determination of Michaelis-Menten Constant7
3.7Ammonium Sulphate Fractionation7
3.8Gel Filtration on Sephadex7
3.9Chromatography on DEAE-cellulose7
4Results8
4.1Freshly Prepared Crude8
4.2Crude Thawed after a Week of Freezing9
4.3Sephadex 25 Gel Filtration11
4.4Determination of Optimum pH12
4.5Ion Exchange Chromatography13
4.6Michaelis-Menten Kinetics13
5Discussion and Conclusion14
65.0 Works Cited14
7Appendix15

Introduction
Acid phosphatases form part of the hydrolase class of enzymes and they can be found in both plant and animal species. Acid phosphatases remove phosphate groups from a broad range of molecules under acidic conditions. (Guo & Pesacreta, 1997). Mobilization of phosphate from organic phosphates present in macromolecules during seed germination and seedling growth are largely carried out by acid phosphatases. (Duff et. al., 1994) In the purification of enzymes, cells must first be disrupted and selectively purified. These steps must be undertaken without a resultant degradation of the enzyme and loss of enzyme activity. Plant acid phosphatases are found predominantly in spereosomes in the cell. Many plant acid phosphatases occur in small quantities with high instability in dilute solutions. These factors, along with the fact that plant acid phosphatases tend to occur in multiple forms makes the isolation of highly purified acid phosphatase increase the chance of denaturation during purification. .Growth of seedlings during germination and functioning of metabolic processes are largely dependent on phosphatase catalysed solubilisation and mobilization of organic sulphates in soils (Bisnol et. al., 1993) There are many variations in isozymes within species. Chromatographic methods have facilitated purification of individual isozymes. It is important to use a standard assay for all activity and concentration measurements. In this paper, the isolation, partial purification and characterization of maize acid phosphatase from maize germ are reported.

Experimental Procedures
Sample preparation
Maize grains of mass 45 g were steeped in water for 48 hours. The water was changed occasionally and placed between two damp cloths and allowed to stand until the maize germinated. Initial Extraction of Acid Phosphatase

All fractionation processes were carried out at 0°C. Seeds which had not germinated were discarded and 40 g of the remainder was blended with an electrical blender. Ice cold water of volume 100ml was added to the crude and the containing vessel was allowed to stand on ice for 30 minutes with stirring. It was then strained with a clean cloth and centrifuged at 10000g for 20 minutes at 0oC and filtered through Whatman No. 1 filter paper. Triton-X 100 of volume 6 ml was added and the sample was divided into two portions. One portion was stored in the fridge and the other sample was stored in the freezer at a temperature of -20oC. Determination of Protein Concentration by the Folin-Lowry Assay To 1 ml of phosphatase solution in a test tube, 5 ml of alkaline reagent was added and mixed. This was left to stand for more than 10 minutes. To the sample in the test tube, 5 ml of Folin-Ciocateau reagent (diluted 1 in 3) was added with rapid mixing. The absorbance was read after 30 minutes at a wavelength of 750 nm. The protein concentration was determined using 0.1% bovine serum albumin (BSA) with a concentrations ranging between 0 and 1 mg/ml. Specific Activity...

Cited: Guo, J.& Pesacreta T.C., (1997). Purification and characterization of an acid phosphatase from the bulb of Allium cepa L. var. sweet Spanish. J. Plant Physiol., 151: 520-527.
Duff, S.M.G., G. Sarath and W.C. Plaxton, (1994). The role of acid phosphatases in plant phosphorus metabolism. Physiol. Plant., 90: 791-800.
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