Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

John B. Schenkman, Ingela Jansson, Yuri Lvov, James F. Rusling, Salah Boussaad, Nongjian J. Tao

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5docking domain. This diminishes the extent of docking of the cytochrome b5In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.

Original languageEnglish (US)
Pages (from-to)78-87
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume385
Issue number1
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

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Keywords

  • AFM of P450
  • P450 sidedness
  • P450 surface charges
  • P450 topology
  • QCM adsorption of P450

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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