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

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