Sequence-specific photomodification of oligodeoxynucleotide pAGAGTATTGACTTA ("a target") has been carried out with the aid of complementary fluorescent probes. Such a probe consisted of oligodeoxynucleotide pAATACTCT and a chromophore group attached to its 5' end. Three different derivatives of ethidium bromide were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2). The irradiation induces the following photodamages: target cleavage at the specific binding site with a cutting off of the 8-mer from its 5' end (yield up to 12%), formation of specific covalent adduct target-probe with a yield of 20-70%, and piperidine-sensitive target modifications with a 7-27% yield (for different chromophores). The total yield of specific photodamages of all kinds is 50-80%. The target cleavage and generation of piperidine-sensitive modifications are optically nonlinear processes. Piperidine treatment of the irradiated samples led to specific cleavage of the target with the yield up to 40%. All kinds of observed modifications are not influenced by high concentrations of free radical scavengers: 1.3M tBuOH and 10 mM cystamine. The pattern of cleavage indicates that the most probable position of the chromophore is between T8 and G9 of the target, i.e., the chromophore stacks on top of the last A.T base pair of the duplex. The aggregate of evidence is in agreement with the mechanism of nonlinear photomodification (the cleavage and generation of piperidine-sensitive modifications) based on the transfer of two-photon excitation energy from the chromophore to the target.

Nonathymidilate was synthesized containing the chromophore (dansyl) group linked to its 5'-phosphate. In the presence of this compound the polyadenilic acid molecules are split by the radiation (power density J t 70 MW/cm2) of a nitrogen laser, while under the same conditions poly(C) and poly(U) are hardly affected. This selective optically non-linear effect was predicted and is explained in terms of radiativeless transfer of two-quantum excitation of the chromophore which is fixed on poly(A) molecule due to the formation of the complementary complex with nonathymidilate. Laser scission Two-quantum excitation Polyadenilic acidComplementary complex of the chromophore Dansyl derivative of nonathymidilate Light-induced diffusion

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For the first time the method of two-quantum affinity modification has been employed to probe structure of an enzyme, bacterial luciferase. Position of the f lavin-binding site of this enzyme , which was previously unknown , has been estabi ished . The obtained data evidence that the f 1 avin site is posit loned on the a-subunit. The most close contact of protein chain of the enzyme with chromophoric group of f 1 avin takes p1 ace near 80± 10 and 120± 10 amino acid residues ; the regions 50± 10 and 215± 10 are a iso c lose to f lavin . The estabi ished localization does not contradict to suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evol utionary stabi 1 ity of various 1 uci ferases . The present method can, in principle, be applied to a great number of enzymes, including all f lavin-dependent enzymes.Enzymatic catalysis has high speed and specif icity [ 1 1 . Creation of a method of determination of the e lements of the primary structure of a protein , making up the active site ( in whi ch substratum conversion occurs) , could be a signif i cant advance in clearing up mechanisms of enzymatic catalysis.It was proposed [ 2 ) to 1 ocal ize active sites of the enzymes , whose substrata are chromophores , using the method of two-quantum aff in ity modif ication . An enzyme-substratum complex is irradiated with laser light of sufficiently long wavelength (X > 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighbouring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site.In the present paper the above approach has been implemented for the first time . Information has been obtained about the posit ion of f 1 avin-binding site of bacterial luciferase.In our experiments we used an enzyme from luminous bacteria Photobacterium leiognathi. It consists of two subunits having molecular weight 41000 and 38000 correspondingly. The enzyme catalyzes the reaction of oxidizing aldehyde and reduced f 1 avin (FMN) [3]. The enzyme specimen was prepared by the mul t istep clearing technique [4]. Protein concentration was measured according to [5). 242 / SPIE Vol. 1525 Future Trends in BiomedicalApplications of Lasers(1991) 0-8194-0653-8/91 /$4.00 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/15/2016 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx