Active Cystatin C (Cys-C)

Cystatin C is a member of family 2 of the Cystatin superfamily. It is involved in processes such as tumor invasion and metastasis, inflammation and some neurological diseases. It inhibits many cysteine proteases such as papain and cathepsins B, H, K, L and S. It is ubiquitous in human tissues and body fluids. A point mutation in the gene coding for the 120 amino acid mature Cystatin C causes a hereditary form of amyloid angiopathy in which the protein variant (Leu68 to Gln) is deposited in the cerebral arteries, leading to fatal cerebral hemorrhage. Cystatin C may have additional clinical applications. For example, it is a good marker for glomerular filtration rate. The activity of recombinant rat Cystatin C was measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC in the assay buffer 50 mM Tris, pH 7.0. Papain was diluted to 500 ug/ml in activation buffer 50 mM Tris, 5 mM DTT, pH 7.0 and incubated at room temperature for 15 minutes. The activated papain was diluted to 100 ug/ml in the assay buffer and 20 ul different concentrations of recombinant rat Cystatin C (MW: 14.93 KD) was incubated with 20 ul 100 ug/ml papain at 37 ℃ for 10 minutes. Loading 50 µL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM substrate. Include a substrate blank containing 50 µL of assay buffer and 50 µL of 200 µM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant rat Cystatin C significantly decreased papain activity. The inhibition IC50 was <80 nM.