Investigation of physicochemical changes to L-asparaginase during freeze-thaw cycling.
L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E. coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles. An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity. A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation. Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes. The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa. A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity. Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments. The shortened monomers then aggregate and precipitate.
Jameel
F
F
School of Pharmacy, University of Connecticut, Storrs 06269, USA.
Bogner
R
R
Mauri
F
F
Kalonia
D
D
D001215:Asparaginase / Q000737:chemistry* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D016974:Dickeya chrysanthemi / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005615:Freezing; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D012967:Sodium Dodecyl Sulfate; D001215:Asparaginase