title: The contribution of ionic interactions to the conformational stability and function of polygalacturonase from A. niger.
abstract: Aspergillus niger produces multiple forms of polygalacturonases with molecular masses ranging from 30 to 60 kDa. The high molecular weight polygalacturonase (61+/-2 kDa) from A. niger possesses a pH optimum of 4.3 and a pI of 3.9. The enzyme exhibited high sensitivity, both in terms of activity and structure, in the pH range of 4.3-7.0. The enzyme was irreversibly inactivated at pH 7.0. The enzyme is predominantly rich in parallel beta structure. There is unfolding of the enzyme molecule between 4.3 and 7.0 resulting in irreversible loss of secondary and tertiary structure with the exposure of hydrophobic surfaces. ANS binding measurements, intrinsic fluorescence and acrylamide quenching measurements have confirmed the unfolding and exposure of hydrophobic surfaces. The midpoint of pH transition for both activity and secondary structure is 6.2+/-0.1. The pH-induced changes of polygalacturonase confirm the role of histidine residues in structure and activity of the enzyme. The irreversible nature of inactivation is due to the unfolding induced exposure of hydrophobic surfaces leading to association/aggregation of the molecule. Size exclusion chromatography measurements have established the association of enzyme at higher pH. Urea induced unfolding measurements at pH 4.3 and 7.0 have confirmed the loss in stability as we approach neutral pH.
authors.affiliation: Department of Protein Chemistry and Technology, Technology Block, Central Food Technological Research Institute, Mysore 570020, Karnataka, India.
authors.forename: T C
authors.initials: TC
authors.lastname: Jyothi
authors.forename: Sridevi Annapurna
authors.initials: SA
authors.lastname: Singh
authors.forename: A G
authors.initials: AG
authors.lastname: Appu Rao
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007525:Isoelectric Focusing; D007700:Kinetics; D011096:Polygalacturonase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014508:Urea / Q000494:pharmacology
chemical_list: D007477:Ions; D006639:Histidine; D014508:Urea; D011096:Polygalacturonase