Complement Factor D (CFD) is a serine protease that catalyzes the initial proteolytic step in the alternative pathway of complement. Expressed in adipose tissue at high levels, factor D is also known as adipsin. It is an exceptionally specific protease and the only known protein substrate is factor B in complex with C3. Factor D protease activity is regulated by reversible conformational changes, which differs from the majority of serine proteases whose regulation involves either activation by processing of the zymogens or inactivation by binding of the inhibitors. Compared to its physiologically important proteolytic activity, factor D has much lower activity toward synthetic peptide substrates. However, thioester substrates have been routinely used for assessing factor D activity. The full-length (amino acid residues 1-263) of rat CFD was expressed which activity was measured by its ability to cleaves a thioester substrate Z-Lys-SBzl?HCl. The reaction was performed in 50 mM Tris, 1 M NaCl, pH 7.5 (Assay Buffer), initiated by addition 50 μL of various concentrations of CFD (diluted by Assay Buffer) to 50 μl substrate mixture of 0.2mM Z-Lys-SBzl?HCl and 0.2 mM DTNB. The final well serves as a negative control with no CFD, replaced with 50 μl assay buffer. Then read in kinetic mode for 5 minutes at an absorbance of 405 nm. The specific activity of recombinant rat CFD is > 10000 pmol/min/μg.