One-step purification of lactoperoxidase from bovine milk by affinity chromatography

ATASEVER A., ÖZDEMİR H., GÜLÇİN İ., KÜFREVİOĞLU Ö. İ.

FOOD CHEMISTRY, cilt.136, sa.2, ss.864-870, 2013 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 136 Sayı: 2
  • Basım Tarihi: 2013
  • Doi Numarası: 10.1016/j.foodchem.2012.08.072
  • Dergi Adı: FOOD CHEMISTRY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.864-870
  • Anahtar Kelimeler: Lactoperoxidase, LPO, Affinity chromatography, Enzyme purification, Inhibition, Kinetics, ANTIBACTERIAL, SYSTEM
  • Atatürk Üniversitesi Adresli: Evet

Özet

Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC50 of 0.848.10(-5) M. The K-i for sulphanilamide was determined to be 3.57.10(-5) M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R-z (A(412)/A(280)) value. The R-z value for the purified LPO was found to be 0.7. Maximum binding was achieved and K-m and V-max values were determined. (C) 2012 Elsevier Ltd. All rights reserved.