Alkaline denaturation and partial refolding of pepsin investigated with DAPI as an extrinsic probe.
The binding parameters of DAPI to porcine stomach pepsin have been described in the previous article in this issue (A. Mazzini et al.). Here we exploit the differences in the spectroscopic (fluorescence and circular dichroism) properties of DAPI bound to either native or alkali denatured pepsin. We follow the kinetics of pepsin denaturation around neutrality (pH range 6.8-7.4), at several phosphate buffer ionic strengths (range 0.02-0.25). The dependence of the apparent dissociation rate constant on pH clearly shows that the rate limiting step follows the dissociation of about three acidic protein residues. The accelerating effect by ionic strength we observed can be accounted for by a simple treatment based on both transition state theory and Debye-Hueckel's limiting law. Furthermore, when a solution of pepsin, rapidly denatured at pH 7, is reacidified to a pH between 4.5 and 5.5, a substantial recovery of protein secondary structure, with no enzymatic activity, is observed, judging by the far UV circular dichroism of the protein. This process of partial refolding can easily be followed using DAPI as an extrinsic reporter group, able to monitor the kinetics of formation and decay of a highly fluorescent intermediate. This process becomes faster at a lower pH, at least in the limited range investigated (pH 4.5-5.5), in which the refolded protein does not aggregate, but, in contrast to unfolding, is almost independent in ionic strength.
Favilla
R
R
Istituto Nazionale di Fisica della Materia (INFM), Dipartimento di Fisica, Università di Parma, Italy. favilla@prix1.fis.unipr.it
Parisoli
A
A
Mazzini
A
A
D000818:Animals; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013552:Swine
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D010434:Pepsin A