E10: Wheat Germ Acid Phosphatase Assay

Temperature , amount of enzyme ,and pH

What factors influence the activity of an enzyme and how (increase or decrease)?

as substrate concentration increases, the rate of reaction increases

Describe how the amount of substrate affects the rate of a reaction.

a. depletion of substrate

b. breakdown of enzyme

c. presence of inhibitors

Explain why reaction rates decline with time and use this information to correctly process the data (by choosing the proper data points to do linear regression)

1. The standard curve of absorbance vs nmole/ml of nitrophenol

2. The amount of nitrophenol produced in the assay vs time

Understand how to construct and read the following two graphs (and be able to critique sample student graphs for errors.):

standard curve

A graph that plots known values which then allows the same properties to be determined for unknown samples by interpolation on the graph.

x = nitrophenol concentration (nmol/ml)y= OD410 absorbance

Describe how the nitrophenol standard curve is generated in this experiment. What parameters are used as X- axis and Y- axis?

4.5

What is the pH at which the acid phosphatase activity is measured?

adding KOH

How is the reaction catalyzed by acid phosphatase stopped?

Standard curve

substrate

nitrophenyl phosphate

Enzyme

acid phosphatase

phosphate and nitrophenyl

product

speed in which the substrate is converted to the enzyme and is proportional

the rate of the reaction in limited by the....

assay vs. time

visualize how the concentration of a substance or the activity of a process changes as time progresses.

purpose of an assay vs. time graph

calculating the amount of product formed over time while the amount of product is increasing in a linear manner

The initial velocity of an enzyme catalyzed reaction is determined by

1. Presence of activators or inhibitors

2. Concentration of substrate

3. Temperature and pH

4. Amount of enzyme

Which of the following influence the rates of enzyme-catalyzed reactions?

nitrophenol concentration

In the standard curve for nitrophenol generated for use in this experiment, what is reported on the x-axis?

absorbance at 410 nm

In the standard curve for nitrophenol generated for use in this experiment, what is reported on the y-axis?