Dear,

we are trying to determine the total acid proteolytic activity of fish extracts. First, we use pepsin as a reference compound and hemoglobin as substrate. According to the procedure, after the reaction the absorbance should be measured at 280 nm. However, we found that at this wavelenght there is too much noice and there is a maximum peak around 400 nm. Is it correct to use this wavelenght instead of 280 nm? Can anyone please help us with this issue?

We use the procedure described below:

Total acid proteolytic activity was determined (triplicate) according to the methodology described by Khaled et al. (2011), with modifcations to substrate concentration, incubation time, temperature and pH of the buffer. A mixture (100mL) containing 2% hemoglobin (w/v) pH 2.5, 50 μL of crude stomach extract, and 350 μL of glycine – HCl 0.1 M buffer pH 2.5 was incubated for 30 min at 37 °C. 500 μL of 10% trichloroacetic acid (w/v) was added after 30 minutes and the mixture was incubated for a further 15 min at 25 °C. The mixture was centrifuged at 10,000 × g for 10 min and the absorbance of the supernatant was analyzed at 280 nm.

Thank you!