Lab #5 info

Lab #5 info - Laboratory V Topic: Enzyme Kinetics...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
Laboratory V Topic : Enzyme Kinetics Principle : All enzymes show distinct kinetic features. During the initial period of an enzyme reaction, there is an excess of enzyme as compared to the substrate. Hence, the reaction velocity increases dependent on the substrate concentration. This is described as the initial velocity . As the reaction proceeds, all the enzyme sites get occupied until the velocity reaches a maximum velocity ( Vmax ). The reaction then continues at a steady state at the maximum velocity. If we plot a graph of the velocity of the enzyme reaction (V 0 ) vs the enzyme substrate [S] using a constant enzyme concentration , it appears as a hyperbola. This plot is mathematically described by Michaelis Menten equation and useful to compare the activity of enzymes. The Km is determined by drawing a Michaelis Menten graph or using the Michaelis Menten equation. The concentration of the substrate that will produce half the Vmax is known as Km . An enzyme with smaller Kms is more active than enzymes with higher Km values. As the substrate is used up, its concentration decreases, and the velocity of the reaction begins to slow down. In today’s exercise, we will look at the kinetics of Acid phosphatase enzyme. Enzyme acid phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate disodium (p-NPPO4 Na 2 ). The enzymatic reaction produces p-nitrophenol (p-NP) and phosphate. The enzyme works optimally in an acidic pH (pH 4.8) and at 37 ° C. Activity 1: Hydrolysis of p-NPP using acid Phosphatase enzyme .You will use a fixed concentration of the enzyme and vary the concentrations of the substrate p-nitrophenyl phosphate. The reaction will be stopped in 30 mins using NaOH. We measure the absorbance of p-nitrophenol formed at 400 nm. Activity 2: a) Standard curve of p-nitrophenol . You will measure the absorbance of p-Nitrophenol at different concentrations and plot a graph of Absorbance (y-axis) vs the concentrations of p-nitrophenol (x-axis) b) Initial velocity of
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 03/09/2011 for the course PHS 2301 taught by Professor Bohu during the Spring '11 term at St. John's.

Page1 / 5

Lab #5 info - Laboratory V Topic: Enzyme Kinetics...

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online