When Lucigenin reacts with hydrogen peroxide in alkaline solutions the major product are postulated to be N-methylacridone. In our studies we investigated the ability of binding of NMA to a serum lipoprotein (BSA). Bovine serum albumin is a relative small protein (MW = 70kd) found in mammalian blood plasma and serum. BSA is a “carrier” protein that helps the distribution of cations and many others insoluble materials such as Ca²⁺, Cu²⁺, hormones and drugs through the body .Understand the interaction of probes with serum proteins components are critical in all biological assay where it is used as a probe.

In all experiments we used a fresh BSA stock solution in saline phosphate buffer (PBS) at 10 mM, pH 7.4. Experiments were made at (37± 1 )°C.The absorption spectra were recorded on a Hitachi U-3000 spectrometer, fluorescence measurement( steady state and time resolved) were monitored on a Hitachi F4500 Spectrofluorimeter FL 900 and Single photon counting respectively.

The quenching of N-alkylacridone by BSA were monitored using excitation in the isobestic absorption region of N-alkylacridone at 372 nm and emission collected in the range from 380 to 550 nm.

Two types of titration were performed:

• titration of different concentrations of BSA protein by successive additions of aliquots of 50mM solution of N-alkylacridone

• titration of saline phosphate buffer solution with 50mM solution of N-alkylacridone.

The Scatchard plot ( Figure 4 ) plots of bound/free vs bound ligant are a common graphical representation of binding data. A Scatchard plot, may be the most common presentation of ligant-acceptor binding data in the biological material. In Figure 4 it was derived from data obtained from Figures 1,2 and 3, shows that there is a single binding site for NMA and NBA, and four binding sites for NDA.In these assay it were also determined K (intrinsic association constant for the binding sites) for both probes, NMA K=0,30x10⁶M^-1 (37⁰C) , NBA K=0,36x10⁶M^-1 (37⁰C) and NDA K = 1,56x10⁶M^-1 (37⁰C)

The quenching of fluorescence in micelle solutions has been studied extensively.We also investigated the distribution for both probes and quenchers in anionic micellar solution of Sodium Dodecyl Sulfate (SDS). In these assay it were avaliated the efficience of quenchers such as Cu²+, MV²⁺ and DoPy⁺ in SDS 40 mM.We obtained quenching constants for NMA ( k_{SV Cu2+} =595M^-1, k_{SV MV2+}=3967M^-1 , k_{SV BeV2+}=766 M^-1 , k_{SV ProPy+}=512 M^-1 and k_{SV DoPy+} = 227M^-1); NBA ( k_{SV Cu2+} = 879 M^-1, k_{SV MV2+}=6168M^-1 , k_{SV BeV2+}= 891 M^-1 , k_{SV ProPy+}=490 M^-1 and k_{SV DoPy+} = 285 M^-1) and NDA ( k_{SV Cu2+} =661M^-1, k_{SV MV2+}=5194M^-1 , k_{SV BeV2+}=892 M^-1 , k_{SV ProPy+}=537 M^-1 and k_{SV DoPy+} = 267M^-1) based in a Stern Volmer formalism. According to these data we propose the possible position of these probes into the micellar aggregates (Figure 6). In BSA, it was postulated that the preferential postion is the one showed in the Figure 7.