Use of substrates with fluorescent donor and acceptor chromophores for the kinetic assay of hydrolases

Abstract

We ~vist/to prer~t a new method for the kinetic study and quantitative assay of hydrolytic enzymes. The method is based on the interruption ofnohradiative ~mergy'transfer between two chromophores attached ioa Subst~te molecule. Enzymic cleavage is followed,.npon excitation of the donor, by moni~o~ dlher the increase.in fluoxesc~rW.e of the donor or the ckcreaie in the fluorescence of the acceptor.Non-radiative energy transfer is a phenomenon in wtdc,~t a.n excited fluorescent ch.romophore (the donor) transfers its/excitation energy to another du~m~hore (the acceptor)i ~ transfer results m ~luench~)g of the fluorescence of the donor ~d appearanoe-ofthe cha~cteristtc fluorescence of the ~p't0r,.althoush the latter is no: excited directly. As ~OWn by FSrstet [! ] the yield of transferdeIs~/ds,'a~on8 oiher factors, on the extent of overlap between the emission spectrtnn of the donor and the ebsorption spectrumof the acceptor, add decreases with thesixth power of the distance between the ch~rom.o .pho.~. Sepdt~atioia dtstan-..es rllowing efficient transfer in known donor.-acceptor pah.; range up to 50A [2--41.-, .• ,.-.-,,-Formula I For compounds in which both cJtromophores are attached to the same molecule, energy transfer at high dilution will be exclusively int~.molecular. Enzymic cleavage of su=h compounds, resulting in the separation of the donor ~nd the acceptor moieties by diffuskm, should obviously lead to a drop in the yield of energy transfer. This effect can be used to follow enzymic activity by monito.~tg either the enhancemerit of fluores~enov of the do~or or the reduction in fluorescence of the acceptor upon exaltation of the donor.• As an illustration to the method we have stu~lied the tryptic digestion of several peptides blocked by the donor 2-naphthyl methyl amide and the acceptor anthra~ne-9-carbonyl amide, Fig, I depic~ the absorption spectrum of anthracene-9-carbonyl-0-alanyllysyl-alar, yl-2-naphthyl methyl amide hydrobromide (I) and the emission spectra of acetyl--lysyl-lysyi-2-naphthyl methyl amide di-hydro-chloride (II) and anthracene-9-cethonyi-~-alarcyl lysyl methyl ester hydrobromide (111). The ahu3rption~p~.~rum of ! is an exact superpmition or" the absorption spectra of ll and HI; at the longer wavelengths (315-400 tun) it is due exclusively to the anthracene moiety, whereas at the shorter wavelengths (270-290 nm) it is .mainly due to the naphthalene moiety. Fig: 1 shows that there is a considerable overlap between the emission band Of the naphthalene donor and the absorption band of the anthracene acceptor. A high yield of energy transfer in o)mEpun d I should therefore be expected. This is clearly si~own in fig. 2, where the emission spectrum of peptide !, due to excitation at 284 rim, is presented. Though at this wavelength the absorption is due almost completely to the naphthalene moieW, its emission is strongly quenchedNorth~Holland Pub~ighing Company '-Amsterdam ! |

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

“…Profluorescent peptide protease detection reagents used in the past have also contained two fluorophores, one on each side of the cleavage site (15,16). However, in contrast to the design presented in the present work where the two dyes are identical, the prior work used peptides with two different, but spectroscopically complimentary, probes (vide infra).…”

Activatable Optical Probes for the Detection of Enzymes

“…The quenched fluorescent signal from the amino acid could be recovered upon hydrolysis with carboxypeptidase A, resulting in a 100-fold increase in fluorescence [3]. Shortly thereafter the first probe synthesized with a non-natural donor and acceptor, napthylene and anthrocene respectively, was reported [4]. Once again cleavage of the dipeptide, in this case by trypsin, resulted in the recovery of the fluorescence emission from the donor.…”

Use of a Fluorescence Plate Reader for Measuring Kinetic Parameters with Inner Filter Effect Correction

“…FRET 2 (fluorescence resonance energy transfer) molecules (1) have been extensively used as substrates for cellulase (2), hydrolases (3,4), and especially proteases (5)(6)(7)(8)(9). The intramolecular FRET protease substrates consist of a peptide with a fluorescence donor at one side of the enzymatic cleavage bond and a quenching acceptor at the other side.…”