Stopped-flow front-face fluorometer: a prototype design to measure hemoglobin R----T transition kinetics

Area of Science:

• Biophysics
• Biochemistry
• Physical Chemistry

Background:

• Hemoglobin's R to T state transition is crucial for oxygen transport.
• Intrinsic and extrinsic fluorescence can report on this transition.
• Stopped-flow techniques enable kinetic studies of rapid biological processes.

Purpose of the Study:

• To develop and validate a stopped-flow front-face fluorometer for kinetic studies of the hemoglobin R to T transition.
• To probe the R to T transition at specific sites using intrinsic and extrinsic fluorescence.

Main Methods:

• Utilized a Gibson-Durrum stopped-flow apparatus integrated with a digital data analysis system.
• Employed front-face fluorometry with narrow band pass filters (470 nm excitation, 520 nm emission).
• Studied the R to T transition by mixing oxy-hemoglobin A labeled with 5-iodoacetamidofluorescein (Hb A-AF) with deoxygenated buffer.

Main Results:

• The developed stopped-flow fluorometer successfully monitored the hemoglobin R to T transition.
• Observed a first-order reaction with a rate constant of approximately 8 s-1.
• This rate constant is consistent with reported oxygen dissociation rates for oxy-hemoglobin A.

Conclusions:

• A stopped-flow front-face fluorometer is a viable tool for studying hemoglobin kinetics.
• The instrument can probe conformational changes like the R to T transition at specific molecular sites.
• The kinetics of the R to T transition can be accurately measured using this technique.