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Fluorogenic oligopeptide derivatives of the type Lys(ABz)-X-ONBzl, where ABz is o-aminobenzoyl (anthraniloyl), X stands for Ala, Phe, or Ala-Ala, and ONBzl is p-nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruption of the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyl ester group. The spectral characteristics of the compounds are not consistent with an energy transfer mechanism according to Forster, therefore the quenching is assumed to be caused by a direct encounter between the quenching and the fluorescent groups. The change in fluorescence that accompanies the enzymic hydrolysis of the first peptide bond was used for quantitative measurement of the activity of leucine aminopeptidase and for the determination of some of its kinetic parameters. A bacterial aminopeptidase from Clostridium histolyticum that is very similar to leucine aminopeptidase in its substrate specificity did not hydrolyze the above peptide derivatives. The hydrolysis of leucine p-nitroanilide by this enzyme was found to be inhibited by the three peptides and the corresponding inhibition constants were determined.Spectrophotometric methods commonly used for assaying the activity of aminopeptidases and for studying their substrate specificity are based on the hydrolysis of p-nitroanilides [l] or P-naphthylamides [2] of amino acids, leucine derivatives being the most widely used. In these substrates the chromophores are directly engaged in the enzymically affected bond, the cleavage of which results in significant spectral changes, due to the conversion of the aromatic amide into an aromatic amine. Since the interaction of aminopeptidases with natural substrates involves the recognition of more than one amino acid residue [3,4], it should be of advantage to develop derivatives of oligopeptides rather than of amino acids as chromogenic substrates. For this purpose, however, a different approach is required that would allow spectrophotometric monitoring of the hydrolysis of an N-terminal peptide bond in spite of the requirement that the chromophore probe cannot be directly engaged in the Ahhrrviations. Abbreviations for amino acid derivatives and peptides follow the recommendations of IUPAC-IUB Commission on Biochemical Nomenclature, see Eur.

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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 ! |

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