Optical biosensor investigation of interactions of biomembrane and water‐soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

• DOI: 10.1002/jmr.532
• Publication Date: 2002-09-10T23:33:12Z
• Authors: Y. D. Ivanov; I. P. Kanaeva; O. V. Gnedenko; V. F. Pozdnev; V. V. Shumyantseva; N. F. Samenkova; G. P. Kuznetsova; A. M. Tereza; R. D. Schmid; A. I. Archakov
• Keywords: Cytochrome b5; Cuvette; Phosphatidylethanolamine; Adrenodoxin; Bovine serum albumin; Protein–lipid interaction

Abstract A phospholipid‐containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor interactions of membrane water‐soluble proteins in cytochrome P450‐containing monooxygenase systems with planary layers dilauroylphosphatidylethanolamine (DLPE) distearoylphosphatidylethanolamine (DSPE), differing acyl chain length. It shown that full‐length proteins—cytochrome P4502B4 (d‐2B4), b5 (d‐b5) NADPH‐cytochrome P450 reductase (d‐Fp)—readily incorporated into phospholipids. The incorporation largely due hydrophobic membranous protein fragments phospholipid layer. However, electrostatic forces were also but not always involved process. They promoted d‐Fp had no effect d‐b5 incorporation. In low ionic strength buffer, these two DSPE lipid layer observable. Incorporation DLPE abruptly increased at temperatures exceeding phase transition point. d‐2B4 dependent its aggregation state decreased increasing aggregability. Water‐soluble either would interact (adrenodoxin) or bind cost only (albumin) both (P450cam) interactions. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: Ad adrenodoxin AS surface biosensor 2B4 cytochromes d‐ t‐ truncated forms 1,2‐dilauroylphosphatidylethanolamine (C12:0) 1,2‐distearoylphosphatidylethanolamine (C18:0) EDC 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide Fp K (Na)P K(Na)‐phosphate buffer KP/E K‐phosphate 0.25 g/l Emulgen 913 NHS N ‐hydroxysuccinimide P450cam PBS/T potassium/Tween