Fluorometric Assessment of Sulfhydryl Oxidase Activity: Optimization by Response Surface Methodology

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Sulfhydryl oxidase activity is implicated in various biological processes.
• Accurate and sensitive assays are crucial for studying enzyme function.
• Existing methods for sulfhydryl oxidase measurement have limitations.

Purpose of the Study:

• To develop and optimize a novel spectrofluorometric assay for sulfhydryl oxidase.
• To evaluate the assay's performance characteristics.
• To apply the assay for determining seminal sulfhydryl oxidase activity.

Main Methods:

• Development of a spectrofluorometric assay utilizing hemoglobin (HB) and hematin (HT) as peroxidase mimics.
• Catalysis of hydrogen peroxide-dependent thiamine oxidation.
• Optimization of the assay using response surface methodology (RSM).

Main Results:

• The assay demonstrated high accuracy, sensitivity, and linearity up to 200 IU.
• Satisfactory correlation was observed when compared to a colorimetric method.
• Reference values for seminal sulfhydryl oxidase activity were determined.

Conclusions:

• The novel spectrofluorometric assay is a precise and sensitive tool for measuring sulfhydryl oxidase activity.
• The use of inexpensive thiamine as a substrate enhances its practicality.
• This method is suitable for evaluating seminal sulfhydryl oxidase in clinical and research settings.