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Enzyme Research
Volume 2012, Article ID 173831, 13 pages
Research Article

Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

1Department of Chemistry, University of Idaho, 875 Perimeter Drive, MS 2343, Moscow, ID 83844-2343, USA
2Department of Biochemistry, University of Nigeria, Nsukka, Enugu State 410001, Nigeria

Received 28 April 2012; Revised 28 July 2012; Accepted 29 July 2012

Academic Editor: Joaquim Cabral

Copyright © 2012 Charles O. Nwamba and Ferdinand C. Chilaka. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant 𝐾 π‘š and 𝑉 m a x values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln ( [ 𝑃 ] ∞ βˆ’ [ 𝑃 ] 𝑑 ) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1 / [ 𝑃 ] ∞ versus 1 / [ 𝑆 ] yielded π‘˜ + 0 and π‘˜ β€² + 0 , the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of π‘˜ + 0 and π‘˜ β€² + 0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.