Dear colleges
I have a small project for identification and determination of aromatic amino acids that may be present in protein hydrolysate of some kind of seeds using spectrofluorometry.
first, we crushed our seeds and separate the oil from it .. after that we make the protein hydrolysis by using different types of enzyme such as pepsin and papain.
My question is : how do we prepare this protein hydrolysate for analysis with spectrofluorometry ? or what is the best reagent that can be used?
please, can anyone give me a suggestions? or any related articles?
best regards
Just dissolve the sample completely in distilled water, pour into a cuvette, and place in the sample chamber of the spectrofluorometer. Do several dilutions and calculate the concentration by averaging, discarding extreme readings. Confirm results by using another solvent, for example, dichloromethane.
8/30/18
Dear Arvan,
A good way to detect amino acids (AA) is with the Roth Reagent. This is orthophthaldialdehyde (OPA). It reacts with primary amines in the presence of a thiol (usually beta-mercapto-ethanol, or BME) to form an iso-indole derivative that is highly fluorescent. The method is extremely sensitive.
Attached are some references describing the use of this reagent. As described in the original paper by Roth, I think borate buffer was used as the sample solvent; however, I don't think this is critical. Other buffers can also be used, e.g., acetate, PO4. DO NOT use Tris (or some other buffer that contains a primary amine), as OPA reacts indiscriminately with any primary amine and gives a reaction, so you will get a very strong reaction with the buffer alone.
The reagent calls for use of BME. If you don't like using BME, you can use another thiol, e.g., N-acetyl-cysteine (NAC) or mercapto-propionic acid. NAC is best, as it is a crystalline solid w/ no odor; it is also a safe reagent to work with.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research (CCUR), Iowa State University, Ames, Iowa, USA [email protected]
You first have to know, what you want to know... Using pepsin and papain you will not get a complete hydrolysis to amino acids. This is usually performed with hydrochloric acid (6 N). If you want to quantify each aromatic amino acid, a separation technique would be desirable. In this special case, even a standard reversed phase HPLC would be sufficient:
https://www.hindawi.com/journals/jaa/2016/7374316/
Article Protein Quantification by Derivatization-Free High-Performan...
Thanks for all of you for answering my question....
For Mr Michael G. Weller, I am trying to quantify (tryptophan, tyrosine and phenylalanine) in the hydrolysate fromed by papain and pepsin using SpectroFluorometry....
Do you have any comments about preparation of hydrolysate by this way ? And how to prepare this hydrolysate for measuring by spectroFluorometry ?
Best regards for all
With these enzymes, you end up with a mixture of peptides and some amino acids. Since fluorescence is highly dependent on the chemical environment, I do not believe that a quantitative determination of these amino acids is possible this way. Do you have any literature reference for your approach?
If you want to read about protein hydrolysis for amino acid analysis, you may have a look in this paper:
Article Hydrolysis and amino acid composition analysis of protein
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