Identification and Mass Spectrometric Sequence Studies of Fragments of L-Asparaginase Produced During Freeze/Thaw Cycling

Abstract

L-Asparaginase isolated from Er. chrysanthemi was found to lose activity upon exposure to consecutive freeze/thaw cycles. The cause(s) for this loss of activity were investigated using multiple techniques. SEC using UV, RI and light scattering detectors and SDS-PAGE indicated that the l-asparaginase molecule fragments upon exposure to repeated freezing and thawing cycles. Following up on this information, mass spectrometry was used to identify the fragments as small peptides of molecular weight 615 Da, 1424 Da and 166 Da. Automated Edman sequencing of the frozen and thawed mixture confirmed the presence of fragments and contributed some sequence information. Mass spectral data and sequence studies of these fragments in conjunction with the known sequence of the molecule placed all the fragments within the last 28 C-terminal amino acids. A study of this region using the published 3 dimensional x-ray crystallographic structure of l-asparaginase revealed that the C-terminal region is exposed and can interact with water. The IBI MacVector™ program “Protein Tool Box” predicted that this region is hydrophilic, has a high surface probability and a strong tendency to interact with water. Both tendencies suggest a potential for bond stress during freeze/thaw cycling. This region is not involved at the catalytic core of the enzyme, but fragmentation in this area may result in unfolding and denaturation of the monomer followed by subsequent aggregation into large, insoluble entities and the loss of enzymatic activity.