Table of Contents 1 Table of Contents 2 2 Introduction 4 3 Experimental Procedures 5 3.1 Sample preparation 5 3.2 Initial Extraction of Acid Phosphatase 5 3.3 Determination of Protein Concentration by the Folin-Lowry Assay 5 3.4 Specific Activity Assay 6 3.4.1 Definition of Enzyme Unit and Specific Activity- 6 3.5 Determination of pH Optimum 6 3.6 Determination of Michaelis-Menten Constant 7 3.7 Ammonium Sulphate Fractionation 7 3.8 Gel Filtration on Sephadex 7 3.9 Chromatography on DEAE-cellulose 7 4 Results 8 4.1 Freshly Prepared Crude 8 4.2 Crude Thawed after a Week of Freezing 9 4.3 Sephadex 25 Gel Filtration 11 4.4 Determination of Optimum pH 12 4.5 Ion Exchange Chromatography 13 4.6 Michaelis-Menten Kinetics 13 5 Discussion and Conclusion 14 6 5.0 Works Cited 14 7 Appendix 15
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.
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.