Development and Validation of Micro-Azocasein Assay for Quantifying Bromelain

Area of Science:

• Biochemistry
• Enzymology

Background:

• Proteolytic enzyme activity is crucial for various biological processes.
• Colorimetric methods using azocasein are established for enzyme evaluation.

Purpose of the Study:

• To develop and validate a micro-assay for quantifying bromelain using azocasein.
• To assess the assay's linearity, precision, accuracy, and robustness.

Main Methods:

• A micro-assay was developed involving azocasein substrate, bromelain test solution, and incubation.
• Reaction termination with trichloroacetic acid, followed by centrifugation and absorbance measurement at 410 nm.
• Assay validation included linearity testing, limit of quantification/detection determination, and robustness checks.

Main Results:

• The assay demonstrated good linearity with serially diluted bromelain (200 µg/mL).
• Limit of quantification and detection were determined as 5.412 µg/mL and 16.4 µg/mL, respectively.
• High precision (RSD < 2.0%) and robustness were observed; dH2O diluent showed higher sensitivity.

Conclusions:

• The developed micro-azocasein assay is a reliable and sensitive method for quantifying bromelain.
• The assay's performance is not significantly affected by wavelength variations (400-450 nm).
• Proteolytic activity of bromelain is enhanced by L-cysteine or N-acetylcysteine.