I prepared a 4% soy protein suspension, adjusted the pH to 8.5, ultrasonicated it for 10 minutes, and then hydrolyzed it with 1% Alcalase. After centrifugation, the supernatant was directly used for the DPPH scavenging activity assay. For the DPPH assay, I mixed 4 mL of 0.1 mM DPPH in 95% methanol with 1 mL of the supernatant (protein or hydrolysate) and incubated the reaction at room temperature for 30 minutes in the dark. DPPH solution was used as the control. During the reaction, I observed colloidal formation in the tubes containing the hydrolysate, which is hindering the absorbance reading. The increased turbidity is leading to higher absorbance values, even though the color intensity has reduced, indicating that the hydrolysate is a better antioxidant than the protein, blank, and control. What could be the reason for this colloidal formation?
Proteins (most probably alcalase and non-digested protein) along with the large peptides are being precipitated by the addition of DPPH in methanol. Most of the proteins do not like organic solvents...Try to apply UF to remove relatively large biomolecules (alternatively apply salting out protein precipitation such as with AS) and test the peptide supernatant for radical scavenging activity...
Thank you for the response İsmail Emir Akyildiz. I have not done ultrafiltration, but I have filtered my supernatant using a 0.45-micron syringe filter, and the results were the same (colloidal formation).
Some proteinaceous substances present low organic solvent solubility (residual proteins, including alcalde, large peptide fragments, and hydrophilic relatively smaller peptides, etc.).
Smaller membrane cut-off values should be preferred. Possibly many hydrophilic compounds along with the protein targets will be prone to be precipitated if the final content of the solution is a high percentage of methanol.
You may apply buffer exchange practices (desalting columns, SEC, UF, Dialysis, resin process, etc...)...Alternatively, you may decrease either methanal or sample volume to observe if any progress on solubility. Then you can find out the sweet spot of the final recipe for the solution of measurement...
Also, the oxidized lipid content of the soybean that is co-extracted during alkaline extraction before hydrolysis should be taken into account, as this can also cause some solubility issues.
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