I am using a fluorescence substrate Abz-12345-DNP for my enzyme to act on it. Where Abz is fluorophore and Dnp is the quencher and 1234 is my amino acid. I just need to know how we can draw a standard for the above activity for the calculation of the amount of product forming.
please provide your valuable input.
The substrate is self-quenched, so it can't be used as is to make the standard curve. There are two solutions. The expensive solution is to synthesize the fluorescent product and use that to prepare the standard curve. The inexpensive solution is to make the product in situ using the enzyme at a concentration sufficient to completely convert the substrate to product in a reasonable amount of time. By using different substrate concentrations and waiting until the reaction has gone to completion, the fluorescence measurements made at the point of completion will be the standard curve.
I use Abz-Phe-Arg-pNA (p-nitroaniline) 1 mg/mL solution with dimethyl formamide. After hydrolysis with NaOH 1M (1:1) during 4 hours pNA concentration can be estimate from absorbance at 405 nm (EM = 8900 M-1). As Abz/:pNA ratio is 1:1 you can estimate the Abz concentration from pNA concentration evaluated on 405 nm.
Initial solution must be diluted to obtain a linear curve.
sir, can we buy Abz separately for the plotting of the standard curve Adam B Shapiro like previously I was using FAM-1234-QXL and they have provided FAM separately for the standard plot curve.
Roseli A Gomes thank you for your suggestion mam, please explain in my case how I have dissolved and make the curve with my substate if you have any idea.
Using Abz by itself would risk a systematic error because the fluorescence quantum yield of Abz attached to the peptide (minus Dnp) may differ from that of free Abz. I have seen this effect with FAM attached to an oligonucleotide, but I don't know whether it also happens with Abz attached to a peptide.
Ortho-aminobenzoic acid, or anthranilic acid, is available commercially.
https://www.sigmaaldrich.com/US/en/product/sial/a89855
However, in your peptide, it is attached to the rest of the peptide by an amide bond through the carboxylic acid moiety, so a more relevant compound is the amide, anthranilamide or 2-aminobenzamide, which is also available.
https://www.sigmaaldrich.com/US/en/substance/anthranilamide1361588686
Professor Luiz Juliano and Maria Aparecida Juliano sinthesize quenched fluorogenic substrates to several enzymes using Abz and EDDnp flanking aminoacid sequences. Several papers were published and synthesis can be viewed there If you need detailed data.
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