Protein Protocols Protein Protocols

500 Smith
Fig. 1 Mechanisms of the cleavage of bonds to the COOH site (scheme A) and to the NH 2
site (scheme B) of aspartyl residues in dilute acid.
3. Incubate at 108°C for 2 h. (See Note 5.)
4. To terminate the reaction, cool and open the hydrolysis tube, dilute the sample with water,
and lyophilize.
4. Notes
1. The bond most readily cleaved in dilute acid is the Asp-X bond, by the mechanism outlined
in Fig. 1(A). The bond X-Asp may also be cleaved, in lesser yields (see Fig. 1[B]).
Thus, either of the peptides resulting from any one cleavage may keep the aspartyl residue
at the point of cleavage, or neither might, if free aspartic acid is generated by a double
cleavage event. Any of these peptides is suitable for sequencing.
2. The amino acid sequence of the protein can affect the lability of the affected bond. Thus,
the aspartyl-prolyl bond is particularly labile in acid conditions (see Note 3). Ionic interaction
between the aspartic acid side chains and basic residue side chains elsewhere in the
molecule can adversely affect the rate of cleavage at the labile bond. Such problems as
these make prediction of cleavage points somewhat difficult, particularly if the protein is
folded up (e.g., a native protein). The method may well prove suitable for use in cleaving
small proteins or peptides, in which such intramolecular interactions are less likely. Nevertheless,
yields are