Evidence for a Non-Linear Relationship Between β-Galactosidase Activity and Enzyme Concentration Due to the Loss of an Activator During Ammonium Sulphate Precipitation

Abstract

It has been observed that the activity of β -galactosidase precipitated with ammonium sulphate drops non-linearly in dilutions of Tris (10mM, pH 8.0). We sought to provide evidence for this trend, henceforth referred to as the “dilution effect,” as well as possible explanations for this phenomenon. We theorized that this dilution effect was due to the possible loss of activators during the precipitation process in the purification protocol employed in a previous experiment. We diluted the ammonium sulphate precipitated enzyme with Tris to reproduce the dilution effect. Next, we compared the rates at different substrate concentration using Tris or an alternate diluent consisting of filtered cell components less than 30,000 MW. Using the rates obtained from these assays, a Lineweaver-Burk transformation of the Michaelis-Menten equation was done to determine V and K. In the ammonium sulphate precipitated enzyme, we discovered that the Max M contents of the filtrate under 30,000 MW increased affinity but caused no significant change in V. We believe that this is evidence of an activator that is less than 30,000 Daltons Max present in the IPTG induced cells of Escherichia coli.