BL 4820 Biochemical Techniques -- Lecture 2 - Characterizing an Acid Phosphatase
4. Acid Phosphatase - Assay System
In Expt #2, you will be analyzing wheat germ acid phosphatase as an example enzyme. The physiological function of this acid phosphatase is to provide inorganic phosphate to the growing wheat seedling during germination. Many different phosphate esters of sugars and substrates are stored in the wheat seed and these need to be hydrolyzed during germination, which makes the carbohydrates available as an energy source and the phosphate to be used as building blocks in making new cells (new RNA and DNA all need phosphate in their backbones).
So while we could use a phosphate ester of a natural compound like glucose-6-P which was discussed in the general discussion of this lecture, it is also possible to use artificial phosphate esters since the enzyme is rather non-specific and will catalyze phosphate ester hydrolysis on many different substrates. Phosphate ester of p-nitrophenol is a good substrate to use since the product formed after ester hydrolysis, p-nitrophenol, can easily be measured.
Here is the reaction catalyzed by wheat germ phosphatase with p-nitrophenylphosphate (pNPP):
The first step is the enzyme catalyzed part of this graphic. In this reaction, the catalyst is the phosphatase and the products are p-nitrophenol and inorganic phosphate or Pi.
The second part of the graphic illustrates the way to detect the p-nitrophenol formed as product in the phosphatase catalyzed reaction shown in part 1. In part 2, NaOH is added to end the phosphatase assay after a given reaction time of say 10 min. The hydroxide reacts with the p-nitrophenol to remove the phenolic proton and p-nitrophenolate is formed, which is a yellow colored compound absorbing at 405 nm. Since nothing much else in the reaction mixture absorbs light at this wavelength, it is easy to measure in a quantitative way the amount of p-nitrophenol formed in the enzyme catalyzed reaction.
This type of enzyme assay is called an "end-point" assay since the activity of the enzyme is evaluated after a given time of reaction. The other type of enzyme assay is a continuous assay where the appearance of product or disappearance of substrate is measured all the time during the reaction. End-point enzyme assays are often used in clinical and other analytical biochemical assays since they are easy to do and often are useful for detecting the presence of an enzyme in a sample in a quantitative manner.
To quantify the amount of p-nitrophenol formed during the phosphatase catalyzed reaction, you need to make a standard curve using known amounts of p-nitrophenol:
You will be provided with a standard solution of p-nitrophenol in 0.02 M NaOH, so it will already be yellow. From this stock p-nitrophenol (pNP) solution, you can prepare various dilutions of p-nitrophenolate (pNP) for constructing the standard curve. Subsequently, when you do a phosphatase assay with pNPP, you can determine the amount of p-nitrophenol made during the reaction time by measuring the A-405 nm and reading the amount of p-nitrophenol from the standard curve. The pNP standard curve should be very linear and so it works well to do a linear regression analysis on it and use the equation of the line to convert the A-405 nm values into amounts of pNP in terms of µmol or pmol.
It may be useful for you to review the concepts of the standard curve and linear regression in Lecture 1 and the Help on Units (last part of this help page).