Peptide Sequencing: Partial Hydrolysis with Cyanogen Bromide: Videos & Practice Problems

Peptide sequencing is a crucial process in biochemistry, and one effective method for achieving this is through partial hydrolysis using Cyanogen Bromide (CNBr). This compound is particularly notable for its specificity; it cleaves peptide bonds exclusively at the carboxyl group of the amino acid methionine. This unique characteristic makes CNBr an invaluable tool in peptide sequencing, as it allows for precise fragmentation of peptide chains without any exceptions.

Understanding the mechanism of CNBr is essential for researchers working with peptides. The cleavage of peptide bonds by CNBr facilitates the analysis of the resulting fragments, which can then be sequenced to determine the order of amino acids in the peptide. This step is foundational in various applications, including protein identification and characterization.

In summary, the use of Cyanogen Bromide in peptide sequencing exemplifies the importance of specificity in biochemical techniques. By targeting methionine residues, researchers can effectively break down peptides into manageable fragments, paving the way for further analysis and understanding of protein structure and function.

In this example, we explore the cleavage of an unknown peptide using cyanogen bromide, which specifically cleaves at the carboxyl side of methionine residues. The absence of methionine-to-methionine peptide bonds indicates that the fragments generated from the cleavage are not directly connected through methionine. This means that one of the fragments can stand alone, while the others must be connected based on the cleavage pattern.

To determine a possible peptide sequence, we first recognize that cyanogen bromide cleaves at the carboxyl group of methionine. Given the fragments, we can deduce that one fragment must be linked to another, while the methionine fragment must be positioned at the end of the sequence. This is because the cleavage occurs on the carboxyl side, meaning that if methionine were at the beginning, it would not allow for a valid connection with the other fragments.

Thus, the proposed peptide sequence can be constructed as follows: starting with methionine, followed by a four-amino acid fragment, and concluding with a three-amino acid fragment. This arrangement respects the cleavage rules and provides a coherent sequence for the peptide chain.

Cyanogen bromide undergoes a partial hydrolysis mechanism that involves a series of nucleophilic substitution reactions. The first step is characterized by an S_N2 reaction, where a sulfur atom, possessing a lone pair of electrons, attacks the carbon atom of cyanogen bromide. This interaction results in the formation of a sulfonium ion as the bromine atom, a good leaving group, departs. The resulting structure is crucial for the subsequent steps.

In the second step, another S_N2 reaction occurs intramolecularly. Here, the carbonyl oxygen attacks the gamma carbon, leading to the expulsion of methylthiocyanate. This reaction facilitates the formation of a five-membered ring structure, which is essential for the final product. The nitrogen atom, with its lone pair, forms a double bond with the carbon, while the oxygen utilizes one of its lone pairs to bond with the carbon, completing the ring formation. The five-membered ring consists of five interconnected atoms, including the alpha, beta, and gamma carbons, along with the carbonyl carbon.

In the final step, acid-catalyzed hydrolysis occurs, cleaving the imine and releasing the methionine derivative from the peptide chain. The presence of a carbon double-bonded to nitrogen signifies the imine, and upon hydrolysis, this bond is broken, regenerating the carbonyl group and introducing an oxygen atom. The nitrogen is protonated, resulting in two hydrogen atoms being added to it. This step effectively separates the PEP_C group from the five-membered ring, which retains the PEP_N group.

Overall, the mechanism of partial hydrolysis by cyanogen bromide involves careful tracking of the nucleophilic attacks and the formation of key intermediates, ultimately leading to the generation of a five-membered ring and the release of specific molecular fragments.