Document Type

Honors Thesis

Publication Date

Spring 2019

Abstract

This thesis research focuses on the interaction of gold(I) thiolates with the disulfide bonds in proteins, using insulin as a model protein. Insulin contains three disulfide bonds that can break apart during thiol-disulfide exchange reactions. The goal of this research was to compare the reactivity of aromatic thiols and gold(I) thiolates in thiol-disulfide exchange reactions with insulin. When the disulfide bonds in insulin are cleaved, a suspension of the beta chain particles forms, which scatters 650 nm light, therefore making it convenient to monitor the reaction using UV-Vis spectroscopy. The rate of formation of the colloidal suspension is taken to be directly proportional to the rate of disulfide bond breakage, as reported by Lees, et al.1 who studied the ability of aromatic thiols to catalyze the reaction of dithiothreitol (DTT) and insulin. The project began by measuring the relative rate of the reaction between 4-mercaptobenzoic acid, DTT and insulin vs DTT and insulin, which was also done by the Lees research group. All reactions were conducted at room temperature in pH 6.5 phosphate buffered solution. The rate of disulfide bind cleavage in the reaction of 4-mercaptobenzoic acid, DTT and insulin was 1.7 times faster than the reaction of DTT and insulin. This is consistent with the relative rate of 1.6 reported by Lees, et al.1 and provided confidence that using the light scattering technique described by Lees, et al. to monitor the reactions with insulin was a viable method in our lab. Another experiment was conducted to see whether 4-mercaptobenzoic acid would react with insulin by itself; however, this reaction did not produce a colloidal suspension within a 24-hour period of time, which suggests that 4-mercaptobenzoic acid does not cleave the disulfide bonds in insulin in the absence of DTT. The reaction between 2-mercaptopyridine (Spy), DTT and insulin was also investigated, and it was determined that 2-mercaptopyridine behaves similarly to 4-mercaptobenzoic acid. The next set of experiments was conducted to determine the effect of replacing aromatic thiols with gold(I) thiolates. Addition of Et3PAu(Spy) to a solution of DTT and insulin caused cleavage of the disulfide bonds in insulin, as observed by an increase of light scattering at 650 nm. The shape of the absorbance (intensity of light scattering) vs. time graph suggests two or more possible competing mechanisms. This is further supported by the observation that Et3PAu(Spy) reacts with DTT and insulin separately. In the reaction of Et3PAuSpy and insulin, there is formation of a colloidal suspension while in the reaction between Et3PAuSpy and DTT, 2-mercaptopyridine is produced, as evidenced by UV-Vis and 1H NMR spectroscopy. The findings of the study suggest that Et3PAu(Spy) undergoes a thiol disulfide exchange reaction with insulin, causing precipitation of the β-chains. Furthermore, this reaction occurs in the absence of DTT, which is different than the reactivity of aromatic thiols. It is not known whether the Et3PAu moiety binds to one of the insulin chains or whether further reaction occurs. Additional studies should be done to deduce the mechanism of the reaction. Et3PAuCl was also shown to react with insulin, resulting in the formation of a precipitate. Since Et3PAuCl cannot undergo thiol disulfide exchange, it is suggested that Et3PAuCl interacts with other donor atoms such as histidine residues in insulin, instead of interacting with the disulfide bonds, but further studies should be done to deduce the mechanism of the reaction.

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