A SERS-Based Analytical Strategy for Real-Time Quantitative Kinetic Analysis and Inhibitor Screening for Cytochrome...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Cytochrome P450 (CYP) enzymes are crucial for drug metabolism and toxicology.
• Real-time monitoring of CYP-mediated reactions is challenging with conventional methods.

Purpose of the Study:

• Develop a surface-enhanced Raman scattering (SERS)-based platform for direct, real-time tracking of CYP enzymatic metabolism.
• Extract critical kinetic parameters and enable inhibitor screening for CYP-mediated reactions.

Main Methods:

• Constructed an in vitro CYP enzyme system using pyrene (Pyr) as a model substrate.
• Utilized cetyltrimethylammonium bromide (CTAB)-induced silver aggregates for simultaneous detection of Pyr and its metabolite 1-hydroxypyrene (1-OHPyr).
• Employed CTAB as an internal standard for quantitative kinetic assessment.

Main Results:

• Achieved real-time monitoring of CYP-catalyzed Pyr oxidation, yielding pseudo-first-order kinetics (k = 0.00501 min⁻¹) and a Michaelis constant (Km) of 13 μM.
• Identified CYP1A2 as the primary isoform for Pyr metabolism through inhibition studies with α-naphthoflavone.
• Demonstrated the platform's capability for label-free, time-resolved enzyme kinetic studies and inhibitor screening.

Conclusions:

• The SERS-based platform offers a novel, simple, and label-free approach for dynamic biochemical analysis of CYP metabolism.
• This method expands SERS applications from static detection to time-resolved enzymatic studies.
• The platform facilitates enzyme kinetic studies, inhibitor screening, and metabolic analysis with high sensitivity and real-time capabilities.