A simple purification procedure of buffalo lung cathepsin H, its properties and influence of buffer constituents on the enzyme activity

• Main Authors: Shalini Singh, Samir Sharma, Sudhir K. Agarwal
• Format: Article
• Language: English
• Published: Elsevier 2020-07-01
• Series: Biochemistry and Biophysics Reports
• Subjects: Buffalo cathepsin H; Lung cathepsin H; Purification; Properties; Buffer constituents
• Online Access: http://www.sciencedirect.com/science/article/pii/S2405580819301955

Background: Cathepsin H (E.C.3.4.22.16) belongs to a family of lysosomal cysteine protease which regulates diverse normal biological processes mainly in intracellular proteolysis. Methods: Purification of cathepsin H from an unstudied system i.e. buffalo lung has been achieved by a simple process developed after incorporating appropriate alteration in the available methods for isolation of the enzyme from other sources. The use of DEAE-Cellulose and SP-Sephadex C-50 helped in better and simultaneous separation of cathepsin B and H up to homogeneity. Results: The SDS-PAGE result showed buffalo cathepsin H to be a single-chain molecule having MW, NH2- and COOH- terminal residues of 25.4 kDa, Lys and Val respectively. The enzyme was a glycoprotein with pI of 6.2; it hydrolyzed Leu-NA (Vmax/Km = 301.6) as the most efficient substrate followed by Arg-NA, Arg-Arg-NA and BANA. Buffalo enzyme showed maximum activity at 36 °C, pH 6.75 and at a buffer concentration of 2 × 10−3 M. Conclusion: Catheptic activity was found to be quite stable at least for 20–30 min between pH 4.5–7.0, buffer concentration of 1 × 10−2 to 4 × 10−2 M and the temperature resistance up to 36 °C. The effects of various substances present in the buffers routinely used for the assay of catheptic activity revealed that the activity of buffalo lung cathepsin H depends not only qualitatively but also quantitatively on the constituents of assay buffer. General significance: This study seems to provide valuable information regarding the biochemistry of cathepsin H in general as well as influence of buffer constituents on enzyme activity and physiological role in particular.