Rapid affinity chromatographic method for the isolation of human cathepsin H.
Cathepsin H was purified by a single-step affinity chromatographic method from crude human kidney extract. The affinity medium consisted of low-molecular-mass cysteine proteinase inhibitors from potato tubers (PCPIs) coupled to cyanogen bromide-activated Sepharose. The yield of the method is comparable to that of the classical methods. Isoelectric focusing and sodium dodecyl sulphate polyacrylamide electrophoresis showed high purity of the isolated cathepsin H. N-Terminal sequence analysis revealed that intact single-chain cathepsin H was obtained. Binding of the enzyme to the PCPI-Sepharose showed that a free SH group in the cysteine proteinase is not required for complex formation.
Popović
T
T
Department of Biochemistry, J. Stefan Institute, Ljubljana, Slovenia.
Brzin
J
J
Ritonja
A
A
Svetic
B
B
Turk
V
V
D000595:Amino Acid Sequence; D056655:Cathepsin H; D002403:Cathepsins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D012685:Sepharose
D015853:Cysteine Proteinase Inhibitors; D012685:Sepharose; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534126:CTSH protein, human; D056655:Cathepsin H