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The influence of ionic strength on the kinetics of selected enzymes.

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pH studies are used to gain insight into chemical mechanisms of enzyme catalysed reactions. However, perhaps the most important practical point that is often overlooked in pH studies is control of the ionic strength of reaction mixtures at the various pH values. For example, cathepsins Band L were suspected to be involved in cancer invasion but pH vs activity profiles indicated that they were not active at the extracellular pH (pH 7.2). When these profiles were re-evaluated in buffers of constant ionic strength, as opposed to buffers of constant molarity, it was shown that the enzymes were indeed active at pH 7.2. Other enzymes have also been reported to be sensitive to ionic strength. These include neutrophil elastase, class sigma glutathione S-transferase and penicillin G-acylase amongst others. The effects of increasing ionic strength on the activity of six enzymes were investigated. a-Glucosidase (from bakers ' yeast), elastase (human leukocyte) and trypsin (bovine pancreatic), cathepsin L (sheep liver), cathepsin B (rabbit liver), fruit bromelain (pineapple fruit) were subjected to different ionic strength buffers and their activities and Km and Vmax were determined as a function of ionic strength. The influence of ionic strength on Ki values has not been previously reported and was also studied, using the interaction between chicken egg-white cystatin C and cathepsin L as a model. a-Glucosidase was found to have an ionic strength optimum and elastase showed increasing activity with an increase in ionic strength. Trypsin activity decreased with increasing ionic strength with a substrate containing a positively charged side chain in the P1 position, and an increase in activity with a substrate containing a hydrophobic group at the P1 position. Cathepsin B activity increased when acting on the substrate Z-Phe-ArgNHMec and decreased when acting on Z-Arg-Arg-NHMec, with increasing ionic strength. Bromelain showed an increase in activity with increasing ionic strength. Cathepsin L activity decreased at increasing ionic strength and the Ki values for the cathepsin L-cystatin C interaction increased with increasing ionic strength. The results obtained can be attributed to the nature of the specificity pockets involved in binding the substrate, effects on the catalytic mechanism of the enzyme or structural changes due to increasing ionic strength. These results show that the ionic strength is a significant variable and should be kept constant or at in vivo levels when assaying enzymes.


Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.


Ionic solutions., Enzymes., Enzyme kinetics., Hydrogen-ion concentration., Buffer solutions., Enzyme inhibitors., Hydrolases., Proteinase., Glucosidases., Ions., Theses--Biochemistry.