title: The Effect of pH on Globular State of Lipase-3646; an Appropriate Model for Molten Globule Investigations.
abstract: Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.
authors.affiliation: Bioprocess Engineering Group, Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Sharek-e Pajoohesh Km 15, Tehran-Karaj Highway, P.O. Box: 14965/161, Tehran, Iran.
authors.forename: Bahram Pooreydy
authors.initials: BP
authors.lastname: Golaki
authors.forename: Saeed
authors.initials: S
authors.lastname: Aminzadeh
authors.forename: Ali Asghar
authors.initials: AA
authors.lastname: Karkhane
authors.forename: Bagher
authors.initials: B
authors.lastname: Yakhchali
authors.forename: Parisa
authors.initials: P
authors.lastname: Farrokh
authors.forename: Ferdous Rastgar
authors.initials: FR
authors.lastname: Jazii
authors.forename: Mohammadsadegh
authors.initials: M
authors.lastname: Nadimifar
mesh_terms: D020106:Acrylamide; D056449:Bacillales / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D020106:Acrylamide; D008049:Lipase