Practical Protein Chemistry - A. Darbre 1989

Chemical fragmentation of polypeptides
Introduction

A. Fontana (A. FONTANA, Institute of Organic

As a rule, the Introduction/19.html">Primary Structure of a protein cannot be determined by sequential sequencing of the entire polypeptide chain. Instead, one must analyze fragments obtained by Selective Cleavage of the polypeptide chain using chemical or enzymatic Methods. Consequently, the fragmentation of the polypeptide chain into low-molecular-weight Peptides for subsequent structural analysis is one of the most critical stages in determining the covalent (primary) structure of a protein.

This chapter discusses Methods for the Chemical Fragmentation of polypeptide chains, while issues related to Enzymatic Hydrolysis are covered in Chapters 3 and 10.

Chemical cleavage methods are based on the ability of chemically modified amino acid residues to activate the peptide bonds of adjacent chain links [20, 21]. Protein Chemistry has utilized cleavage at specific amino acid residues for many years. A practical method for the selective chemical cleavage of polypeptide chains must meet two key requirements: high selectivity and a high yield. Ideally, the cleavage of a specific peptide bond should occur without any side reactions. In practice, however, known chemical cleavage methods are not without limitations; foremost among these are low reaction yields and undesirable side reactions.

One of the primary requirements for a fragmentation technique is high Specificity—that is, the cleavage of only a few polypeptide bonds, resulting in a peptide mixture that is relatively easy to separate. Although it is theoretically possible to determine a fairly long Amino Acid Sequence by automated sequencing, it is much more practical to obtain a small set of large fragments that, following Separation, serve as more manageable targets for automated sequencing. Potential targets for chemical Reagents aimed at limited fragmentation are the Functional groups of relatively rare Amino acids, such as Methionine, Tryptophan, and Cysteine.

The demand for methods to produce large fragments has stimulated growing interest in The Development of specific cleavage techniques. This chapter focuses primarily on those methods that have been validated and applied in practice. Alongside well-established methods of cleavage at methionine, tryptophan, Tyrosine, and cysteine residues, this chapter also examines Partial Acid Hydrolysis, as well as reductive and oxidative cleavage at cystine residues. The literature on the chemical fragmentation of Peptides and Proteins is extensive, so the authors have not attempted to cover every aspect of the subject. Readers seeking a more in-depth exploration of this topic can find comprehensive information in detailed reviews [4, 74, 78, 81, 83, 181, 209].