title: Conformational changes of a chemically modified HRP: formation of a molten globule like structure at pH 5.
abstract: Horseradish peroxidase is an all alpha-helical enzyme, which widely used in biochemistry applications mainly because of its ability to enhance the weak signals of target molecules. This monomeric heme-containing plant peroxidase is also used as a reagent for the organic synthesis, biotransformation, chemiluminescent assays, immunoassays, bioremediation, and treatment of wastewaters as well. Accordingly, enhancing stability and catalytic activity of this protein for biotechnological uses has been one of the important issues in the field of biological investigations in recent years. In this study, pH-induced structural alterations of native (HRP), and modified (MHRP) forms of Horseradish peroxidase have been investigated. Based on the results, dramatic loss of the tertiary structure and also the enzymatic activity for both forms of enzymes recorded at pH values lower than 6 and higher than 8. Ellipticiy measurements, however, indicated very slight variations in the secondary structure for MHRP at pH 5. Spectroscopic analysis also indicated that melting of the tertiary structure of MHRP at pH 5 starts at around 45 °C, which is associated to the pKa of His 42 that has a serious role in keeping of the heme prostethic group in its native position through natural hydrogen bond network in the enzyme structure. According to our data, a molten globule like structure of a chemically modified form of Horseradish peroxidase at pH 5 with initial steps of conformational transition in tertiary structure with almost no changes in the secondary structure has been detected. Despite of some conformational changes in the tertiary structure of MHRP at pH 5, this modified form still keeps its catalytic activity to some extent besides enhanced thermal stability. These findings also indicated that a molten globular state does not necessarily preclude efficient catalytic activity.
authors.affiliation: Department of Biology, Payame Noor University (PNU), Tehran, Iran.
authors.forename: Kourosh
authors.initials: K
authors.lastname: Bamdad
authors.affiliation: Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-154, Iran.
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.affiliation: Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-154, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Naderi-Manesh
authors.affiliation: Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-154, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Sadeghi
keywords: conformational transition; horseradish peroxidase; molten globule like structure