solutionsch05 - 2608T_ch05sm_S54-S62 2/1/08 5:56PM Page...

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Protein Function chapter 5 S-54 1. Relationship between Affinity and Dissociation Constant Protein A has a binding site for ligand X with a K d of 10 ± 6 M. Protein B has a binding site for ligand X with a K d of 10 ± 9 M. Which protein has a higher affinity for ligand X? Explain your reasoning. Convert the K d to K a for both proteins. Answer Protein B has a higher affinity for ligand X. The lower K d indicates that protein B will be half-saturated with bound ligand X at a much lower concentration of X than will protein A. Because K a ² 1/ K d , protein A has K a ² 10 6 M ± 1 ; protein B has K a ² 10 9 M ± 1 . 2. Negative Cooperativity Which of the following situations would produce a Hill plot with n H ³ 1.0? Explain your reasoning in each case. (a) The protein has multiple subunits, each with a single ligand-binding site. Binding of ligand to one site decreases the binding affinity of other sites for the ligand. (b) The protein is a single polypeptide with two ligand-binding sites, each having a different affinity for the ligand. (c) The protein is a single polypeptide with a single ligand-binding site. As purified, the protein preparation is heterogeneous, containing some protein molecules that are partially denatured and thus have a lower binding affinity for the ligand. Answer All three situations would produce n H ³ 1.0. An n H (Hill coefficient) of ³ 1.0 gener- ally suggests situation (a) —the classic case of negative cooperativity. However, closer exami- nation of the properties of a protein exhibiting apparent negative cooperativity in ligand bind- ing often reveals situation (b) or (c). When two or more types of ligand-binding sites with different affinities for the ligand are present on the same or different proteins in the same solution, apparent negative cooperativity is observed. In (b), the higher-affinity ligand-binding sites bind the ligand first. As the ligand concentration is increased, binding to the lower-affinity sites produces an n H ³ 1.0, even though binding to the two ligand-binding sites is completely independent. Even more common is situation (c), in which the protein preparation is heteroge- neous. Unsuspected proteolytic digestion by contaminating proteases and partial denaturation of the protein under certain solvent conditions are common artifacts of protein purification. There are few well-documented cases of true negative cooperativity. 3. Affinity for Oxygen of Hemoglobin What is the effect of the following changes on the O 2 affinity of hemoglobin? (a) A drop in the pH of blood plasma from 7.4 to 7.2. (b) A decrease in the partial pres- sure of CO 2 in the lungs from 6 kPa (holding one’s breath) to 2 kPa (normal). (c) An increase in the BPG level from 5 m M (normal altitudes) to 8 mM (high altitudes).
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This note was uploaded on 05/20/2011 for the course BCH 3218 taught by Professor Johnsteward during the Spring '08 term at University of Florida.

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solutionsch05 - 2608T_ch05sm_S54-S62 2/1/08 5:56PM Page...

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