title: Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris).
abstract: The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221 005, UP, India. shyama@csb.sunysb.edu
authors.forename: Shyamasri
authors.initials: S
authors.lastname: Biswas
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010835:Phytohemagglutinins / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D005456:Fluorescent Dyes; D010835:Phytohemagglutinins; D010940:Plant Proteins; C101135:phytohemagglutinin L protein, Phaseolus vulgaris; D019791:Guanidine