Two-state irreversible thermal denaturation of anionic peanut (Arachis hypogaea L.) peroxidase

Published Date
Thermochimica Acta
9 July 2004, Vol.417(1):67–73, doi:10.1016/j.tca.2004.01.018

• Author 

• Laura S. Zamorano ^a
• David G. Pina ^b
• Francisco Gavilanes ^c
• Manuel G. Roig ^a
• Ivan Yu. Sakharov^d
• Andrei P. Jadan ^e
• Robert B. van Huystee ^f
• Enrique Villar ^b
• Valery L. Shnyrov ^b,,

• aDepartamento de Quı́mica Fı́sica, Facultad de Quı́mica, Universidad de Salamanca, 37008 Salamanca, Spain
• bDepartamento de Bioquı́mica y Biologı́a Molecular, Universidad de Salamanca, 37007 Salamanca, Spain
• cDepartamento de Bioquı́mica y Biologı́a Molecular, Facultad de Quı́mica, Universidad Complutense, 28040 Madrid, Spain
• dDepartment of Chemical Enzymology, Faculty of Chemistry, Moscow State University, 118899 Moscow, Russia
• eInstitute for Biological Instrumentation of the Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
• fDepartment of Plant Sciences, The University of Western Ontario, London, Ont., Canada N6A 5B7

Received 20 August 2003. Revised 19 January 2004. Accepted 19 January 2004. Available online 5 March 2004. 

Abstract

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the thermal stability of anionic peanut peroxidase (aPrx) at pH 3.0. The spectral parameters were seen to be good complements to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. The results, supported with data concerning changes in enzymatic activity with temperature, show that thermally induced transitions for aPrx are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of aPrx denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme,  , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Keywords

Irreversible thermal denaturation

Differential scanning calorimetry

Intrinsic fluorescence

Circular dichroism

Peanut peroxidase

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