The prominent conformational plasticity of lactoperoxidase: A chemical and pH stability analysis

Barbara Boscolo, Sónia S. Leal, Carlos A. Salgueiro, Elena Maria Ghibaudi, Cláudio M. Gomes

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective Cm values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

Original languageEnglish
Pages (from-to)1041-1048
Number of pages8
JournalBiochimica Et Biophysica Acta-Proteins And Proteomics
Volume1794
Issue number7
DOIs
Publication statusPublished - Jul 2009

Keywords

  • Chemical denaturation
  • Heme protein
  • Mammalian peroxidase
  • pH effect
  • Protein folding
  • Structural stability

Fingerprint

Dive into the research topics of 'The prominent conformational plasticity of lactoperoxidase: A chemical and pH stability analysis'. Together they form a unique fingerprint.

Cite this