PARTITION EFFICIENCY OF HIGH-PITCH LOCULAR MULTILAYER COIL FOR COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF PROTEINS USING SMALL-SCALE CROSS-AXIS COIL PLANET CENTRIFUGE AND APPLICATION TO PURIFICATION OF VARIOUS COLLAGENASES WITH AQUEOUS-AQUEOUS POLYMER PHASE SYSTEMS

Abstract

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially higher stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, IV, V, and X derived from Clostridium histolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample. Collagenase from C. histolyticum well retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.

Keywords:

• collagenases
• countercurrent chromatography
• cross-axis coil planet centrifuge
• high-pitch multilayer coil
• locular tubing
• polymer phase system
• proteins
• purification

ACKNOWLEDGMENT

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The authors would like to thank Mr. Kazunori Yoshida and Mr. Kazuhiro Yanagidaira (College of Science and Technology, Nihon University, Chiba, Japan) for their technical assistance. The authors are also indebted to Mr. Susumu Kasahara (Takacho Co., Tokyo, Japan) for his helpful advice.

Notes

Abbreviations: Cyt C = cytochrome C; Myo = myoglobin; Lys = lysozyme. The average of partition coefficient (K) values (C_U/C _L ) for each protein were 0.11 for Cyt C, 0.56 for Myo and 1.66 for Lys, respectively.

Abbreviations: Lys = lysozyme; Myo = myoglobin. The average of partition coefficient (K) values (C_L/C_U) for each protein were 0.64 for Lys and 1.90 for Myo, respectively.

Distribution ratios were calculated from the absorbance of the upper phase (C _U ) divided by that of the lower phase (C_L) obtained by the spectrophotometric method at UV 280 nm.

*Protein concentrations were calculated from the assumed absorbance at 1.0 (280 nm) of 0.1% protein solution.