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Lecture 2

CHEM564 Lecture 2: Case study.2

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Christopher Cairo

BioconjugateChemistry Cairo 6.1.2 Detection of free thiols in a protein Determination of the oxidation state of a protein can be important for a variety of applications. For proteins with a large number of Cys residues, oxidation to disulfides could alter protein structure or function. Moreover, in a case with more than one Cys there are a number of degenerate oxidation states. In this example, we will examine a straightforward strategy for detecting the range of oxidation states populated by a protein containing multiple Cys residues using SDS-PAGE and bioconjugate chemistry. Creighton and coworkers were interested in the analysis of bovine pancreatic trypsin 9 inhibitors (BPTI). The protein is ~6.5 kDa, and contains six Cys residues. At the time of these studies, there were few tools for microanalysis of protein samples to determine individual species. Instead, analysis of aminoacid content by protein sequencing would provide an average number for each residue. For example, if a sample contained 50% of four reduced Cys and 50% of three reduced Cys the determination would be 3.5 reduced Cys. Thus, one is unable to distinguish the case above from a situation with 50% of five and 50% of two reduced Cys, or other degenerate cases. Creighton et al. developed a simple method to resolve this problem by altering the properties of the BPTI analyte. The reduced Cys residues retain their activity as nucleophiles, and this can be exploited by reaction with -bromoketones. Reaction with iodoacetic acid or iodoacetamide (Scheme IV) will convert the thiols to thioethers. This transformation has the added benefit of altering the isoelectric point (pI) of the resulting conjugate. For example, reaction with iodoacetic acid should shift the pI lower. Reaction with the i
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