Date of Award

6-1991

Document Type

Open Access

Degree Name

Bachelor of Science

Department

Chemistry

Language

English

Abstract

Intrinsic protein fluorescence has been used to monitor the guanidine HCI induced denaturation of E. coli aspartate transcarbamylase (ATCase) and its isolated catalytic subunit (CSU). ATCase is a large oligomeric protein which contains both regulatory subunits and catalytic subunits. Both tryptophans in ATCase are located in a single globular domain of the catalytic subunit. For both ATCase and CSU, increasing concentralions of guanidine HCI caused a red shift of the maximum protein emission (Aex 295 nm) of 15.0 +/- 0.5 nm. CSU showed a concommitant decrease in fluorescence intensity while ATCase showed an increase from 0.0 to 2.0 M and then a decrease from 2.0 to 4.0 M. For CSU, the equilibrium denaturation profile monitored by emission peak shift showed a highly cooperative but asymmetric transition. This indicated the presence of two transitions, one occuring from 0.0 to 1.75 M guanidine HCI and another occuring from 2.0 to o.O M guanidine HCI. ATCase gave a denaturation profile which was nearly superimposable on that of CSU, except early in the denaturation (0-1.75 M guanidine HCI). This indicated that association of RSU with CSU affected the tryptophan fluorescence of CSU and that RSU has fully dissociated from CSU at a guanidine HCI concentration of 1.75M. Plots of -lnK versus guanidine HCI concentration for both CSU and ATCase gave linear plots, despite the noticeable asymmetry in their denaturation profiles, indicating that this is not an accurate analysis for judgement of the complexity of the denaturation process. The effects of PALA (a bisubstrate analogue of ATCase) on the denaturation profile of CSU and A TCase were also evaluated .

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In Copyright - Educational Use Permitted.