Figure 3. Isolation and purification of calcium binding peptides. (a) Elution profile of BCC hydrolysate by hydroxyapatite chromatography. HA column (20 × 80 mm) was pre-equilibrated with 10 mM of potassium phosphate buffer, pH 6.5. The separation was performed with a linear gradient of 10–200 mM of phosphate buffer at a flow rate of 1.0 ml/min; (b) Elution profile of the active fraction (H4) from HA chromatography further purified by Sephadex G-25 chromatography. The column (26×150 mm) equilibrated and eluted with distilled water at a flow rate of 1.0 ml/min. (c) Elution profile of the active fraction (S3) from Sephadex G-25 chromatography further purified by RP-HPLC. The semipreparative RP-HPLC column (9.4 × 250 mm, Agilent Zorbax SB-C18) was eluted with a linear gradient of 5%–25% acetonitrile in water at a flow rate of 0.5 ml/min. All peaks eluted were monitored at 220 nm and collected for the calcium-binding activity analysis. All data were expressed as mean values (mean ± SD, n = 3)

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Purification of Bovine Bone Oligophosphopeptide with High Calcium-binding Activity by Bacillus cereus MBL13 Collagenolytic Protease

Lili Liu, Chenliu Yang, Yang Zhu, Yuanyuan Meng, Xiaoning Dai

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