[High performance liquid chromatography of fluorescent insulin derivatives]. / Vysokoéffektivnaia zhidkostnaia khromatografiia fluorestsentnykh proizvodnykh insulina.

The reversed-phase HPLC of porcine insulin modified by fluorescent labeling with dansyl chloride, fluorescein isothiocyanate, and an N-hydroxysuccinimide ester of 3-carboxyl derivative of Nile Red was studied. Mono-, di-, and tri-Dns-insulins (substituted at residues Gly1 of the A-chain and Phe1 and Lys29 of the B-chain), as well as isomeric 5'- and 6'-fluorescein thiocarbamoyl-Phe1 insulin derivatives were separated on the analytical and semipreparative scale. The results were interpreted in terms of conservation of the globular structure in the modified proteins and their surface-mediated interaction with the reversed-phase sorbent. Observed retention times correlated with the total hydrophobicity of the surface region containing the incorporated label (in the case of monosubstituted derivatives) or with the total hydrophobicity of chromatographic contact regions located between labels (in the case of di- and trisubstituted derivatives).

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers.

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.