Cooperative Binding: In-Class Demonstration
This page simulates the outcomes of O2 binding experiments by human hemoglobin (Hb A). Background on O2 by Hb can be found in the Lecture 13 notes. Briefly, the binding of O2 can be monitored by the changes that occur in the visible light absorption (410 nm) of the heme prosthetic group. This is a very sensitive signal that allows the use of very low concentrations of Hb A. The Mr of the Hb A tetramer is 72,000 Da.
- You have a 7.2 mg/mL solution of Hb A.
You can add any concentration of the O2 to the cuvette.
- a) Use the resulting values of [O2]bound to calculate Y and [O2]free;
- b) Plot the data on a Hill plot;
- c) Determine Kd and nH, the Hill coefficient;
- d) Submit your data, calculations, and the graph that yields Kd and nH.
(Each calculated [O]bound value has a small "experimental error" added to it.)
Use the steps described on Topic #3: Cooperative Binding Calculations to solve this problem.
Hint: 1. Calculate the concentration of Hb in mM units before starting your data collection. This will enable you to plot Y vs. [O2]free as you get the data.
2. For your submitted graph, ignore the data <10% and >90% saturation when drawing a line. These values will have more relative error and they are not necessary to determine an accurate slope and intercept on the Hill plot.
Print an Answer Form for submitting your solution.
|
Back to the Calculations & Graphing Index