E10: Acid Phosphatase Enzymme Assay Introduction

What are functions of KOH in the assay?

1) stop the reaction by raising the pH to stop the acidic enzyme from performing 2) visualize the product because KOH turns product from colorless to yellow so the product can be used to measure in the spectrophotometer

How do you produce experimentally the product vs time graph?

After performing assay, find absorbance of each A1-A6 and using equation from standard curve, determine concentration of nitrophenol at each time interval.

How do you construct and read the graph for amount of nitrophenol produced in the assay vs time?

After performing assay, find absorbance of each A1-A6 and using equation from standard curve, determine concentration of nitrophenol at each time interval. Plot points and do linear regression. Slope of linear is reaction rate

How do you produce experimentally the standard curve?

Have different concentrations of nitrophenol using serial dilution and read absorbances of each, plot points for graph

How do you construct and read the standard curve of absorbance vs nmol/mL of nitrophenol?

Have different concentrations of nitrophenol using serial dilution and read absorbances of each, plot points for graph. Lineear line represents relation between concentration and absorbance

How was the assay performed?

Have vials A*, A1=A6 1) add 1mL of KOH into A1-A6 2) add 7mL of substrate, pH 4.5, into A* 3) add enzyme to A* and shake 4) transfer 1mL into A1 from A* 5) remove 1mL into A2-A6 at diff times

How do you describe the reaction catalyzed by acid phosphatase?

Substrate nitrophenyl, colorless in alkaline, is catalyzed by acid phosphatase to produce phosphate and nitrophenol, which is yellow in alkaline solutions. KOH stops reaction and provides visual

What is purpose of product vs time graph?

To determine the reaction rate for the acid phosphatase/ the reaction

What is purpose of the standard curve?

To generate equation for relating absorbance and concentration

How do you determine Vo and the equation?

Use the product formed nmoles vs time min graph and use the linear portion of the graph, Vo = slope Equation: product formed = Vo * time

How does amount of enzyme affect the rate of a reaction?

assuming large excess of substrate, reaction rate will proportionally increase as enzyme concentration increases

Why do reaction rates decline with time?

substrate limitation and/ or cofactor depletion

What factors influence the activity of an enzyme?

temperature, pH, amount of enzyme, substrate concentration, and effects of inhibitors and/ or activators

How correctly process data to do linear regression?

use the data points that are initially linear, not the declined part that is due to substrate limitation and cofactor depletion

How does the amount of substrate affect the rate of a reaction?

will increase reaction rate until Vmax reached at point of saturation of all enzyme ports - Km = Vmax/2 approximately and required for effective catalysis, can compare various enzyes to same ssubstrate for affinity