Bilirubin binding with liver cystatin induced structural and functional changes.
Cysteine proteinases and their inhibitors play a significant role in the proteolytic environment of the cells. Inhibitors of cysteine proteinases regulate the activity of these enzymes helping in checking the degdration activity of cathepsins. The bilirubin secreated by liver cells can bind to cystatin present in the liver resulting in its functional inactivation, which may further lead to the increase in cathepsins level causing liver cirrhosis. In case of some pathophysiological conditions excess bilirubin gets accumulated e.g. in presence of Fasciola hepatica (liver fluke) in mammals and humans, leading to liver cirrhosis and possibly jaundice or normal blockade of bile duct causing increased level of bilirubin in blood. Protease-cystatin imbalance causes disease progression. In the present study, Bilirubin (BR) and liver cystatin interaction was studied to explore the cystatin inactivation and structural alteration. The binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of BR with buffalo liver cystatin (BLC). Stern-Volmer analysis of BR-BLC system indicates the presence of static component in the quenching mechanism and the number of binding sites to be close to 1. The fluorescence data proved that the fluorescence quenching of liver cystatin by BR was the result of BR-cystatin complex formation. FTIR analysis of BR-Cystatin complex revealed change in the secondary structure due to perturbation in the microenvironment further confirmed by the decreased caseinolytic activity of BLC against papain. Fluorescence measurements also revealed quenching of fluorescence and shift in peak at different time intervals and at varying pH values. Photo-illumination of BR-cystatin complex causes change in the surrounding environment of liver cystatin as indicated by red-shift. The binding constant for BR-BLC complex was found to be 9.279 × 10(4) M(-1). The cystatin binding with bilirubin has a significant biophysical and pathophysiological significance, hence our effort to study the same.
Department of Biochemistry, Faculty of Life Sciences, A.M.U., Aligarh, 202002, UP, India.
Mir Faisal
MF
Mustafa
Bilqees
B
Bano
D000818:Animals; D001663:Bilirubin / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002020:Buffaloes; D002942:Circular Dichroism; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D015891:Cystatins; D001663:Bilirubin