Resistance of bromelain to SDS binding.
Interaction of the plant cysteine protease bromelain with SDS has been studied using CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe pyrene, isothermal calorimetric (ITC) investigations and inhibition of hydrolyzing activity. Results exhibit number of synchronous transitions when plotted against the total SDS concentration. SDS at submicellar level caused conformation change of bromelain leading to a stable entity. ITC and pyrene experiments suggest that the structural modifications below 5 mM, the cmc(app) of SDS solutions containing bromelain, are the result of alterations of solvent hydrophobicity or non-specific weak binding and/or adsorption of SDS monomers. Melting temperature (T(m)) and the free energy change for thermal unfolding (DeltaG(unf)) of the SDS induced conformers was decreased by 5 degrees C and 0.5 kcal/mol respectively, compared to native bromelain. Below 5 mM, SDS caused large decrease in V(max) without affecting K(m) for the substrate Z-Arg-Arg-NHMec. Analysis of kinetic data imply that SDS acts as a partial non-competitive inhibitor since even at 100 mM, the residual activity of bromelain was retained by 3%. Inhibition studies show an IC(50) of 0.55 mM and a high K(i) of 0.145 mM. These demonstrate that bromelain is resistant to SDS binding and denaturation, a property known for beta-sheet rich kinetically stable proteins.
Bhattacharya
Reema
R
Division of Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India.
Bhattacharyya
Debasish
D
D001963:Bromelains / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D003627:Data Interpretation, Statistical; D007700:Kinetics; D016018:Least-Squares Analysis; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D011721:Pyrenes; D012967:Sodium Dodecyl Sulfate; D001963:Bromelains; C030984:pyrene; C091760:stem bromelain