The effect of ligand binding on protein association

Tellam, Ross (1980). The effect of ligand binding on protein association PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland. doi:10.14264/uql.2014.502

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Author Tellam, Ross
Thesis Title The effect of ligand binding on protein association
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.502
Publication date 1980
Thesis type PhD Thesis
Supervisor D. J. Winzor
Total pages 220
Language eng
Subjects 0601 Biochemistry and Cell Biology
Formatted abstract
This thesis commences with a resumé of binding theory for protein (acceptor) systems in which the addition of a small molecule (ligand) effects a change in the macromolecular state of the acceptor. Then follows a brief description of the diverse experimental systems that have been investigated, together with the rationale for their inclusion in, and relevance to, a study devoted to ligand-mediated changes in protein self-association.

Chapter 2 describes an investigation of the effect of Zn(II) ion upon the macromolecular state of bacterial α-amylase, a study which disproves the published postulate that dimerization of the enzyme in the presence of the metal ion reflects the formation of a zinc bridge between two monomers. Sedimentation studies have established that the enzyme from Bacillus subtilus undergoes reversible dimerization in the absence of Zn(II) ion, and equilibrium dialysis has demonstrated a dependence of the zinc-binding curve on the concentration of a-amylase used in the experiment. These results, which are totally inconsistent with the crosslinking concept of dimerization, are described very satisfactorily by a reaction scheme in which zinc binds exclusively to a single site on the dimer, which coexists in association equilibrium with monomeric a-amylase.

Quantitative studies of the binding of N-acetyl-ʟ-tryptophan to the coexisting monomeric and dimeric forms of α-chymotrypsin are presented in Chapter 3. Sedimentation equilibrium in the absence of the competitive inhibitor has been used to establish the existence of the monomer-dimer equilibrium, and gel chromatography to establish the preferential binding of N-acetyl-ʟ-tryptophan to monomeric α-chymotrypsin. The association constant for the binding of the ligand to monomer has been determined from enzyme kinetic studies of the competitive inhibition, whereas that describing the binding of N-acetyl-ʟ-tryptophan to dimeric α-chymotrypsin has been deduced from gel chromatographic studies. The present results, which indicate that monomer exhibits a five-fold greater affinity for N-acetyl-ʟ-tryptophan than does dimer, refute earlier claims for the exclusive binding of competitive inhibitors to monomeric enzyme, and also those for equivalence of inhibitor-binding to monomeric and dimeric forms of α-chymotrypsin.

Chapter 4 is concerned with apparent inconsistencies in the literature concerning the macromolecular state of bovine neurophysin II. Sedimentation equilibrium experiments have shown that the seeming discrepancy reflects a difference between the neurophysin systems studied. In acetate buffer neurophysin II comprises an equilibrium mixture of monomeric and dimeric forms, but phosphate buffer with similar pH stabilizes the dimeric entity to the extent that the monomer-dimer equilibrium is no longer detected. The implications of this difference in the quaternary state of neurophysin under the two sets of conditions are discussed in relation to the nature of the macromolecular interactions that are responsible for the cooperative binding of the hormone oxytocin to neurophysin.

Analysis of binding results obtained with proteins undergoing reversible self-association requires prior knowledge of the polymerization pattern, which involves the identification of all polymeric species present and also the determination of the equilibrium constants that inter-relate the concentrations of these oligomers. Chapter 5 is concerned with such quantitative characterization of the self-association of α-chymotrypsin under conditions (pH 7.9, I 0.03) where the polymerization pattern is much more complex than the simple monomer-dimer system pertaining at pH 4 (Chapter 3). Quantitative analysis of sedimentation velocity and sedimentation equilibrium experiments on di-isopropyl phosphoryl-chymotrypsin has led to a postulated polymerization pattern which involves isodesmic, indefinite association of dimer, that is formed by the discrete association step observed under conditions where higher polymers are not detected.

Two contributions to the development of methodology for the study of macromolecular interactions are presented in Chapter 6. A simple method is described for determination of the asymptotic boundary shape from a series of schlieren patterns taken during a sedimentation velocity experiment on a rapidly and reversibly associating solute. The form of the boundary so obtained reflects effects of sedimentation and chemical reaction, but is free from diffusional effects. The procedure is illustrated with analyses of experiments on di-isopropyl phosphoryl-chymotrypsin in 0.29 I phosphate, pH 7.9 (a monomer-dimer system) and on β-lactoglobulin A in 0.1 I acetate, pH 4.65 (a monomer-dimer-trimer-tetramer system). Asymptotic patterns so obtained by this empirical procedure exhibit close agreement with those predicted by Gilbert theory.

The second methodological aspect considered in Chapter 6 is the extent of osmotic shrinkage that occurs in chromatographic studies of self-associating proteins on cross-linked polyacrylamide (Bio-Gel) gels. Consideration of the consequences of this osmotic effect is shown to lead to better quantitative descriptions of the concentration-dependences of elution volume observed in gel chromatographic studies of α-chymotrypsin and zinc-free α-amylase respectively. Indeed, for the latter system allowance for the osmotic phenomenon was required in order to obtain even a qualitative indication of the existence of the monomer-dimer equilibrium that has been studied quantitatively by sedimentation equilibrium in Chapter 2.


Winzor, D.J., Tellam, R., and Nichol, L.W. (1977). Determination of the asymptotic shapes of sedimentation velocity patterns for reversibly polymerizing solutes. Arch. Biochem. Biophys. 178, 327-332. doi:10.1016/0003-9861(77)90200-4

Tellam, R. and Winzor, D.J. (1977). Self-association of α-chymotrypsin at low ionic strength in the vicinity of its pH optimum. Biochem. J. 161, 687-694.

Tellam, R., Winzor, D.J., and Nichol, L.W. (1978). Stabilization of the dimeric form of bacterial α-amylase by zinc (II) binding. Proc. Aust. Biochem. Soc. 11, 7.

Tellam, R., Winzor, D.J., and Nichol, L.W. (1978). The role of zinc in the stabilization of the dimeric form of bacterial α-amylase. Biochem. J. 173, 185-190.

Tellam, R., de Jersey, J., and Winzor, D.J. (1979). Evaluation of equilibrium constants for the binding of N-acetyl-ʟ-tryptophan to monomeric and dimeric forms of α-chymotrypsin. Biochemistry (in press). DOI:10.1021/bi00591a009
Keyword Protein binding
Ligand field theory

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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