Adsorption and conversion of prothrombin on a rotating disc

Area of Science:

• Biochemistry
• Surface Science
• Biophysics

Background:

• Protein adsorption rates are typically site-dependent in flow systems, complicating surface-mediated conversion studies.
• The Michaelis-Menten equation is often used to describe protein conversion, but accurate application requires accounting for transport limitations.
• Uniformly accessible surfaces are crucial for quantitative analysis of surface-bound enzyme kinetics.

Purpose of the Study:

• To quantitatively analyze prothrombin conversion by the prothrombinase complex on a uniformly accessible surface.
• To develop a rotating disc system adapted for ellipsometric measurement of protein adsorption and surface-mediated reactions.
• To determine the intrinsic kinetic parameters of prothrombinase activity under controlled transport conditions.

Main Methods:

• Utilized a rotating disc electrode system for uniform surface accessibility.
• Employed ellipsometry to measure protein adsorption kinetics (lysozyme, prothrombin, fibrinogen).
• Assembled the prothrombinase complex on a phospholipid bilayer and measured prothrombin conversion rates at varying surface concentrations and rotation speeds.

Main Results:

• Demonstrated agreement between experimental and theoretical adsorption rates for lysozyme, prothrombin, and fibrinogen.
• Observed that at high prothrombinase surface coverage, conversion rate is transport-limited.
• Estimated intrinsic kinetic parameters (Km = 7.1 ± 1.2 nM, kcat = 25 ± 1.0 s⁻¹) at low prothrombinase concentrations, independent of rotation speed.

Conclusions:

• The rotating disc system provides a reliable platform for studying surface-mediated enzyme kinetics.
• Intrinsic kinetic parameters of prothrombinase are measurable and not significantly affected by fluid shear rate at low enzyme surface densities.
• This methodology allows for accurate kinetic analysis of surface-bound enzymes, overcoming limitations of site-dependent adsorption.