A sensitive chemiluminescence method based on a luminol-myoglobin system is proposed for the determination of melamine in milk products. It was found that the mixed solutions of melamine and myoglobin could react to form a complex on line, which could greatly inhibit the chemiluminescence intensity generated from the reaction between luminol and myoglobin. The decrease in chemiluminescence intensity was proportional to the concentration of melamine, giving a calibration graph linear over the concentration from 10 pg mL(-1) to 50 ng mL(-1) (R(2) = 0.9988) with a detection limit of 3 pg mL(-1) (3sigma). At a flow rate of 2.0 mL min(-1), one analysis cycle, including sampling and washing, could be accomplished in 20 s with a relative standard deviation of <4.0%. The proposed method was applied successfully to the determination of melamine in milk products, and the recovery was from 93.4 to 106.5%. The possible mechanism of luminol-myoglobin-melamine reaction is given.

The photochemical reaction mechanism of lysozyme with cephalosporin analogues was investigated with luminol used as a luminescence probe by flow injection chemiluminescence. It was found that Glu35 and Asp52 of lysozyme accelerated the rate of excited 3-aminophthalate electrons transferring and enhanced the chemiluminescence signal of luminol, producing steady-state chemiluminescence in the flow injection system with relative standard deviations less than 3.0%. It was also found that cephalosporin analogues could enter into the site of Trp62 in lysozyme forming 1 : 1 complex which leads to a conformational change of lysozyme, giving the effect of chemiluminescence quenching from luminol-lysozyme. Based on the photochemical behavior of luminol/lysozyme and cephalosporin, a model of lysozyme-cephalosporin interaction, lg[(I(0)-I)/I]=lgK(D) + nlg[D], was established. Using the proposed model, the interaction parameters and the binding ability of lysozyme with cephalosporin were successfully obtained, and the results agreed very well with the results obtained by fluorescence.

It was first found that the intrinsic fluorescence of lysozyme at 340 nm can be quenched by cephalosporin analogues through the static quenching and non-radiative energy transferring procedure. In the acetate buffer solution with pH 7.0 and 298 K, the quenching fluorescence intensity was in a good linearity over the concentration of drugs in the range of 1-100 micromol L(-1), 0.1-100 micromol L(-1), 0.5-100 micromol L(-1) and 0.05-100 micromol L(-1) for cefradine, cefuroxime, cefotaxime and ceftriaxone, respectively. The quenching ability or the binding ability of the studied drugs followed the pattern: ceftriaxone > cefotaxime > cefuroxime > cefradine, which was close to the order of their antibacterial ability. The binding parameters including the association constant and the number of binding potential point were calculated at different temperatures (288, 298 and 308 K), and thermodynamic parameters DeltaH degrees, DeltaS degrees and DeltaG degrees were given. The binding mode of lysozyme with cephalosporins showed that the hydrophobic effect might play a major role. The binding distance between cephalosporin and tryptophan residue in lysozyme was obtained. The results provided the quantitative information for the binding of cephalosporin to lysozyme, and it was suggested that the drugs probably bound to the active site near Trp62 in lysozyme.