14] Cleavage at Asn[sbnd]Gly bonds with hydroxylamine

Abstract

This chapter presents a procedure for nonenzymic cleavage of proteins with hydroxylamine, which provides a relatively specific means of producing large peptide fragments suitable for further chemical analysis. Cleavage occurs at Asn-Gly bonds and results from the tendency of the asparaginyl side chain to cyclize, forming a substituted succinimide that is susceptible to nucleophilic attack by hydroxylamine. The increased susceptibility of Asn-Gly bonds in comparison with other asparaginyl bonds may result from the greater ease with which the asparaginyl side chain can cyclize in the absence of steric hindrance imposed by a side chain on the succeeding amino acid. The extent of cleavage achieved varies with the protein; cleavage is enhanced by complete denaturation of the protein and by the use of 6 M guanidine as a solvent during hydroxylaminolysis. A low level of cleavage at Asn-X bonds has been observed in some cases, but aspartyl bonds appear to be resistant under conditions used. The infrequency of Asn-Gly bonds in most proteins results in the production of very large fragments that may overlap CNBr-produced fragments and could serve as new start points for sequential Edman degradation.