Ligand Binding - Experiment 19 Ligand Binding of Avidin and...

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: Experiment 19 Ligand Binding of Avidin and Biotin Biochemistry 332L - - 4 2 09 , Having finished with enzyme interactions this week you will apply that , . , knowledge to a very similar topic ligand binding For this lab you will take , a hand in designing the experimental procedure yourself so read the . following and make yourself up a plan for lab on Thursday , This writeup is a bit long because you need something more on paper . to refer to for this lab than for the others we ve done this semester The , , material presented her however is largely just an adaptation of things you , . have seen before when we studied enzyme kinetics and equlibrium systems , Many binding interactions between proteins and small molecules or , between two different proteins are very similar to the binding of enzymes to - their substrates but we will now look at binding that occurs without . chemical catalysis These binding interactions are found in signal , , , transduction gene regulation transport processes allosteric regulation of , . enzymes and many others The binding of two biomolecules can be analyzed by methods that are . familiar to you by now Let us imagine a protein binding to a small organic . ”. molecule This molecule will be called the ligand If the protein were an , enzyme catalyzing a reaction with the molecule we would refer to that . molecule as a substrate of the enzyme But here we are imagining that the , . protein is just binding to it so we are calling it a ligand instead Instead of , ”. the term enzyme we will refer to the binding protein as the receptor ( ) Realize that it is possible for a single protein molecule or an oligomer to . , bind to more than one molecule of the ligand Really we want to think of the receptor as being a single binding site . on the protein One protein ”, molecule might well correspond to two or more receptors as we will use . , , . the term here This in fact is one of the things we will want to determine We can write an association equation for the ligand and receptor as . follows + R L ⇌ LR So , [ ] , [ ] Here L represents the concentration of free ligand R represents the , [ ] concentration of free receptor and LR represents the concentration of the . complex in which ligand and receptor are bound to each other As we ve , seen before where most chemists would look at the above equation and k 1 k -1
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, write the equilibrium constant for the forward direction biochemists instead tend to use the dissociation , constant K d , shown above for the reverse direction in which the bound complex dissociates into free ligand and . receptor Recall that the free energy for a reaction can be calculated from the equilibrium constant from the equation . , In this week s lab we will be examining the binding of the small
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Ligand Binding - Experiment 19 Ligand Binding of Avidin and...

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