Practical 3

EXPLORING A DIAGNOSTIC ENZYME ASSAY

What the practical is about

  • We explore the enzyme phosphatase as part of phosphate cycling (a key characteristic of cellular metabolism). It helps remove inorganis phsophate. This is important in energy transfer as its found in many metabolic compounds.

  • There are 2 primary phosphate found in organisms: acid and alkaline phosphatase. For the cowpea, their acid phosphatase has optimal activity at pH 5.

  • A plant called cowpeas have high levels of phosphatase in their roots, making them grow better. We will take two extracted samples of the phosphatase from two different cultivators and determine which has higher phosphatase activity. We will then conclude which would better tolerate phosphorus starvation.

  • We will explore the ability of phosphatases to dephosphorylate p-nitrophenyl phosphate (PNPP) to p-nitrophenol (PNP). This reaction is used to detect and measure both acid and alkaline phosphatase activity.

  • This is usefull because PNPP is mostly colourless, whereas PNP is yellow when reacting with NaOH. When PNPP (substrate) is placed in the presence of the phosphatase, yellow PNP product will be produced. Amount of product = proportional to enzyme present. A plate reader will then be used. The more yellow, the more light it can absorb. PNP max absorbance is at 405 nm.

3.1

  • Measuring the absorbance of PNP solutions of known concentrations to generate a PNP standard curve.

  • Making a graph of the PNP concentration vs absorbance at 405 nm.

3.2

  • Measuring the absorbance of PNP produced in your cowpea samples but do not know the concentration of the PNP

  • Make graphs again for absorbance in the samples

3.3

  • Using the PNP standard curce to help calculate the concentration of PNP generated by the action if the enzyme phosphatase extracted from your cowpea samples. Then, calculate the rate of PNP production for each cowpea sample which in turn will help determine the level of phosphatase activity in each cowpea cultivator.

  • We use our graphs from 3.2 and our equation from our 3.1 graph to find the PNP concentration.

  • Then calculate the rate of PNP production in both samples using this equation: Rate of PNP production (x 10^-6 M/minute) = [PNP tube 2] - [PNP tube 1] / time of reaction (mins)