Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

Area of Science:

• Biochemistry
• Neuroscience
• Analytical Chemistry

Background:

• Tyrosine hydroxylase (TH) is a critical enzyme in catecholamine synthesis.
• Accurate measurement of TH activity is essential for neurological research.
• Existing radioassays have limitations in sensitivity and sample requirements.

Purpose of the Study:

• To develop a highly sensitive and reliable assay for tyrosine hydroxylase (TH) activity.
• To enable precise quantification of enzymatically formed DOPA.
• To facilitate TH activity measurement in limited biological samples.

Main Methods:

• Development of a novel assay using high-performance liquid chromatography (HPLC) with amperometric detection.
• Implementation of a double-column procedure for rapid isolation of DOPA.
• Utilized Amberlite CG-50 and aluminum oxide columns for DOPA adsorption and elution.

Main Results:

• The assay demonstrated superior sensitivity compared to traditional radioassays.
• Quantification of as little as 5 pmol of enzymatically formed DOPA was achievable.
• Successfully measured TH activity in small quantities (2 mg) of human putamen tissue.

Conclusions:

• The developed HPLC-based assay provides a highly sensitive method for measuring TH activity.
• This technique is particularly suitable for analyzing TH activity in small brain nuclei from human and animal samples.
• The assay offers a valuable tool for neurochemical research and diagnostics.