mpact of Glycosaminoglycans (GAGs) on Stability and Kinetics of Bone Morphogenetic Protein-2 (BMP-2): A Biophysical Approach

Abstract

Bone morphogenetic protein-2 was overexpressed, purified and refolded using gradient dialysis. The stability and kinetics of the BMP-2(bone morphogenetic protein-2) was studied in presence and absence of glycosaminoglycan (Hyaluronic acid and chondroitin sulphate). The experiments are carried out using UV-Vis spectrophotometer, spectrofluorimeter in both wavelength scan and time scan modes, circular dichroism. The kinetics mode was used at 37 ̊C to mimic the cellular environment. Absorbance data and fluorescence intensity confirmed the quenching in both CS ( chondroitin sulphate)1 and HA (hyaluronic acid). The secondary structure of the BMP-2 was significantly changed upon interaction with both HA and CS which introduced more compactness, confirmed by circular dichroism data. Unfolding kinetics of BMP-2 in presence of CS confirmed that the rate of association decreased in presence of 15μM urea. Further the unfolding kinetics of BMP-2 in presence hyaluronic acid and sulphated hyaluronic acid using urea (6M and 8M) suggested an opposite trend. In presence of sulphated HA the rate of unfolding decreased on increasing concentration of HA (0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml.) and the opposite trend was observed in HA on increasing the concentration (0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml.). This study indicates the role of different glycans in proper functioning of BMP-2 can be helpful for further in vivo studies