Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of protein interactomics. This work is a logical continuation of the cycle of our previous works devoted to the study of PPIs among the components of cytochrome P450-dependent monooxygenase system. Using an optical biosensor of Surface Plasmon Resonance (SPR biosensor), a comparative analysis on the determination of kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 with cytochrome P450s was performed using two different protocols for protein immobilization: 1) covalent non-oriented one on to the carboxymethyl dextran chip type CM and 2) non-covalent oriented immobilization in the lipid environment on the chip type L1 with internal control of liposomes surface distribution. In the second protocol it was shown that the complex formation was characterized by 2.5 times higher affinity due to an decrease in rate dissociation constants. The appropriateness of using both experimental models is discussed.
Vyiavlenie novykh belok-belkovykh vzaimodeĭstviĭ (BBV) i kharakteristika kolichestvennykh parametrov kompleksoobrazovaniia predstavliaet soboĭ odnu iz tsentral'nykh zadach belkovoĭ interaktomiki. Dannaia rabota iavliaetsia logicheskim prodolzheniem tsikla nashikh rabot, posviashchennykh issledovaniiu BBV sredi belkov tsitokhrom R450-zavisimoĭ monooksigenaznoĭ sistemy. S pomoshch'iu opticheskogo biosensora poverkhnostnogo plazmonnogo rezonansa (SPR biosensor) byl vypolnen sravnitel'nyĭ analiz kineticheskikh i ravnovesnykh parametrov kompleksoobrazovaniia membrano-sviazannogo gemoproteina tsitokhroma b5 s tsitokhromami P450 v dvukh raznykh variantakh immobilizatsii belkov: 1) kovalentnaia neorientirovannaia immobilizatsiia na chipe s karboksilirovannym dekstranom; 2) nekovalentnaia orientirovannaia immobilizatsiia v lipidnom okruzhenii. Bylo pokazano, chto vo vtorom sluchae BBV kharakterizovalis' bolee vysokoĭ affinnost'iu (v srednem v 2,5 raza) vsledstvie umen'sheniia skorosti raspada kompleksov.
Keywords: biosensor; cytochrome P450; cytochrome b5; liposomes; optical chip CM5 and L1; protein-protein interactions; surface plasmon resonance.