Purification Techniques and Quantitative Analysis of
Alkaline Phosphatase

SDS Gel with standards and purified fractions of alkaline phosphatase. 12/04/12

Laura Walberg
Eastern Connecticut State University
Low Rise 121
83 Windham Street
Willimantic, CT 06226 (203) 947-6311 walbergl@my.easternct.edu Abstract Alkaline Phosphatase is found in the periplasmic membrane of E. coli as a means of inorganic phosphate by cleaving phosphoryl groups such as PNPP. The initial velocity and concentration of PhoA within E. coli can be determined by isolation of the enzyme and then tracking the reactions by absorbance at 410 nm. Alkaline Phosphatase is commonly removed from the periplasmic membrane by lysis and transformation by heat shock. The protein may be further isolated by fractionation with ammonium sulfate and DEAE anion-exchange chromatography. These purification procedures were performed to quantitatively examine the presence of alkaline phosphatase at different fractions throughout protein isolation. A SDS Gel and Western Blot confirmed the purity of the protein, which was determined to have a molecular weight of 64,595 Daltons (Table 5, Figure 6, & Appendix 16) by analyzing the enzyme fractions compared to the standard on the SDS gel with Kodak MITM.

Introduction The purpose of this experiment was to isolate, purify, and characterize the periplasmic enzyme, alkaline phosphatase, in order to create a standard curve to determine its molecular weight. Alkaline Phosphatase catalyzes the reaction R-O-PO3H- +H2O  R-OH +H2PO4- at an optimum pH of 8, as a way of producing inorganic phosphate to the surrounding cell. The hydrolysis of PNPP by PhoA can be followed at 410 nm to calculate the concentration of PNP being produced. This enzyme is dimeric, with a combined molecular weight of 98,000 Daltons; it is only active when paired. Periplasmic proteins make up 10-15% of the total proteins found in E. coli (Ames et al. 1984), therefore multiple steps must be performed to isolate a single enzyme. In this experiment, the membrane was opened and proteins were released into solution using Bio-Rad Quantum Prep®. Studies have also shown that using chloroform to shock the cells is an efficient way to release proteins into solution from the periplasmic membrane (Ames et al. 1984). An agarose gel was run after the initial cell lysis to confirm that the plasmid containing PhoA, which is about 5,000 base pairs, was present, and viewed by BioRad Fast Blast DNA Stain. To further purify alkaline phosphatase, lysozyme can be added to cleave the cell wall. Adding EDTA allows the solution to remove bound calcium ions from the cell wall, therefore softening it. Dialysis is also a crucial step in protein purification; it allows for the extraction of free calcium ions and other solutes and equilibrates with the surrounding buffer, while keeping the proteins intact. Unlike many proteins, PhoA is very heat stable. Heat denaturation removes heat sensitive proteins from solution, and further isolating PhoA. Salting-out with Ammonium Sulfate is also a very common procedure in that it creates a good crude cut for purification, but it is time consuming and may risk damaging the sample. DEAE-cellulose anion-exchange chromatography is useful in a number of ways. Along with fractioning proteins, the solution eluted from the column can be visually analyzed for the concentration of protein present and the amount of enzymatic activity present. This can be done by performing spot tests with Bradford reagent and PNPP, respectively. Bradford reagent is used to estimate the concentration of protein present based on the appearance and intensity of a color change to blue when sample is added. PNPP is used to estimate the ability of a column fraction to hydrolyze PNPP to PNP, indicating that alkaline phosphatase is present. Activity by PhoA is determined based on the appearance and intensity of a color change to yellow