Dear Dr.Udenigwe,

Thank you for your information. I'm comparing the hydrolysis of respective protein via soluble enzyme and immobilized enzyme in a flow-through system , while the latter is impossible to be watched via pH-stat method, this is the reason whey I tried OPA method. Could you give me some advice on this matter? such as some other methods.

Hello Dr.Udenigwe,

Yes, I find the same problem with hydrolysates generated via the soluble enzyme, the absorbance of adducts from different hydrolysates and OPA does not increase with increasing hydrolysis degree (according to the pH-stat method), since I read many papers in which the authors compare pH-stat, OPA and TNBS methods.  At least they are comparable, but it's not in my case. I took samples out at time intervals (such as 5min, 10min, 30min......until 3h), and the max DH is around 5%, so I'm wondering that it's because of the low hydrolysis degree.  Here I attach the detailed OPA method I used, hope there is no mistake......

OPA reagent (≈5.96 mM)

2 ml   80 mg OPA (134.14 g/mol) dissolved in 2ml ethnol

0.2 ml   ß-mercaptoethanol

5 ml  20% SDS

50 ml  Sodium tetraborate buffer (0.1 M, pH9.7-10)

Dilute with distilled water to 100 ml

Tips: Covered with aluminum foil to protect from light and prepared daily

substrate concentration is around 5%, the aliquots taken out was diluted to 10-flod with distilled water then was used for OPA measurement.

20 ul sample + 2.4 ml OPA reagent

the absorbance was read at 340 nm immediately for 3min (data was recorded every 10 s), the absorbance always increases firstly and then followed by a decrease.

  I used OPA method for degree of hydrolysis sodium caseinate, but for some hydrolysates, the absorbance is  lower than samples without hydrolysis. 

to determine the highest possible DH, sodium caseinate was hydrolysed in 6 N HCL for 24 h at 110 C but its absorbance was very low.

please advise me to find the reason.

Hi Yuhong, did you later find out the reasons for the problem? I used the same OPA reagents (Church et al., 1983) as yours but used 400 ul sample + 3 ml OPA reagent for the measurement. I also noticed the absorbance at 340 nm did not increase for the samples with a longer hydrolysis time while SDS-PAGE gel results showed the proteins of these samples had more hydrolysis.

Thanks.