The interaction behavior of bovine serum albumin (BSA), lysozyme (LYS), myoglobin (MB), and catalase (CAT) with luminol, respectively, was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studied proteins can enhance the CL intensity of luminol. A FI-CL model of protein-luminol interaction, , was constructed, and the interaction parameters of BSA, LYS, MB, and CAT with luminol were determined accordingly. The binding constants are in the descending order of CAT > MB > LYS > BSA at the level of 105 to 107？L？mol？1, and the number of binding sites of luminol to BSA or LYS is around 2 and to MB or CAT is around 1. The results of thermodynamic parameters ( , , and ) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force. 1. Introduction Proteins possess many biological functions including binding, catalysis, operating as molecular switches, and serving as structural components of cells and organisms [1, 2] and play an important role in the transportation and deposition of various endogenous and exogenous substances [3, 4]. In recent years, the interaction of protein with small molecules has become a hot spot in the fields of chemistry, biology, and medicine [5–7]. As common model proteins, bovine serum albumin (BSA) , lysozyme (LYS) , myoglobin (MB) , and catalase (CAT)  are widely applied to the study of protein-small molecule interaction. Accordingly, many methods have been utilized to investigate this hot topic, such as fluorescence spectroscopy , resonance light scattering , circular dichroism , nuclear magnetic resonance spectroscopy , and chemiluminescence (CL) with flow injection (FI) technique . The FI-CL analysis has proven to be a very useful analytical method with advantages of simple apparatus, high sensitivity, wide dynamic ranges, reproducibility, automatability, less reagent consumption [17–19], and so forth. Luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione) is known to produce CL with the characteristic wavelength of 425？nm under alkaline condition [20, 21], making it widely used around the world as a blood enhancement technique in forensic science. Also this luminescent compound is used clinically in the treatment of alopecia , the promotion of blood clotting , and wound healing . Due to its high quantum yield, the CL of luminol has been used in many laboratory applications, including environmental field of analyzing trace metals [25, 26], food safety field , biological
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