Question

Which of the following situations would produce a Hill plot with n_H < 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 1

all three situations would produce nH <1.0.

An nH (Hill coefficient) of , <1.0 generally suggests situation

(a) the classic case of negative cooperatively.

However, closer examination of the properties of a protein exhibiting apparent negative cooperatively in ligand binding often reveals situation (b) or (c).

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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 cooperatively is observed.

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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 nH 0<1.0, even though binding to the two ligand-binding sites is completely independent. Even more common is situation

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(c), in which the protein preparation is heterogeneous. 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

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