Study On The Interaction Of Model Proteins With Drugs By Molecular Spectrometry

Protein is the essential biological material and basilic studying object of life science. Study on the interaction between protein and small molecules has been the current important subject in the fields of biology, chemistry and clinical medicine as it can help to explore the relativity of the structure and nature of small molecules with structure and function of protein. In this thesis, based on the CL of luminol can be significantly enhanced by lysozyme, BSA, catalase and myoglobin, the steady CL system of luminol-lysozyme, luminol-BSA, luminol-catalase and luminol-myoglobin was proposed. Using lysozyme and BSA as the model proteins, the interaction of protein with cephalosporin was investigated by FI-CL method and a FI-CL model for studying the interaction of protein-drug was constructed for the first time. The proposed model was applied successfully to the interaction study of four model proteins with drugs. The main content consisted of two parts:Part I:ReviewThe current development of molecular spectroscopy was summarized and the research status for the interaction between protein and small molecules by molecular spectroscopy were described.282 references was cited (in Chapter 1).Part II:Research reports1. Constructing of FI-CL model for studying the interaction of protein-drug. Lysozyme (or BSA) and luminol could react to form 1:1 complex on line, which could greatly enhance the luminol CL intensity, then the steady CL system of luminol-lysozyme (or luminol-BSA) was proposed. Based on cephalosporin binding to luminol-lysozyme (or luminol-BSA) formed 1:1:1 complex and remarkable quenched the CL of luminol-lysozyme (or luminol-BSA), the FI-CL model of protein-drug interaction was constructed and the formula lg(Io-I)/I=lgKD+nlg[D]was deduced (in Chapter 2 and 3).2. Applications of FI-CL model for studying the interaction of protein-drug. Using the proposed model, the interaction parameters of lysozyme-cephalosporin and BSA-cephalosporin were calculated. Meanwhile, the binding parameters were also studied by fluorescence quenching method. The results obtained by the proposed model agreed well with the results obtained by fluorescence quenching method illuminated that the proposed model would be a feasible method in the study of protein-drug interaction. The binding constant KD values of the drugs mostly were at 103-105 level suggesting that there was high binding affinity of cephalosporin to lysozyme or BSA. The binding ability of the studied cephalosporin drugs followed the pattern:cefoperazone, ceftriaxone and cefotaxime> cefuroxime and cefaclor> cefadroxil, cefradine and cefazolin, which consisted with that of their antibacterial ability. The number of binding potential point n approximately equaled to 1.0 indicating that there was one class of binding site to cephalosporin analogues in lysozyme or BSA and cephalosporin probably bound to the site of Trp62 in lysozyme or the active site near Trp212 in BSA. The calculated thermodynamic parameters were△G＜0,△H>0 and△S> 0, which meant that the binding process was spontaneous and the hydrophobic effect was the major binding force in the interaction of lysozyme or BSA with cephalosporins (in Chapter 2 and 3). Based on the inhibitory effect of cephalosporin on the luminol-catalase CL system, a new method for studying the interaction of catalase with cephalosporin was constructed. Utilizing the proposed protein-drug interaction model, the binding constant and the number of binding potential point were calculated. The results indicated that there was high binding affinity and one class of binding site of cephalosporin in catalase. The calculated thermodynamic parameters showed that catalyse bound with cephalosporins by hydrophobic effect. The proposed procedure was applied successfully to determine cefradine in capsules and monitor the excretion of cefradine in human urine samples. It was found that the excretive cefradine concentration reached its maximum after orally administrated for 2 hours, the cefradine excretive ratio in 8 hours was 79.51%, the total elimination rate constant (K) and the half-life time (t1/2) of cefradine in the body were 0.4822 and 1.45, respectively (in Chapter 4). Based on the inhibitory effect of cephalosporin on the luminol-myoglobin system, the interaction of myoglobin with cephalosporin was studied by FI-CL and the interaction parameters were obtained. The proposed method was applied successfully to the determination of cefaclor in capsules with the recoveries range from 93.0%-106.5%, and the results agreed well with the labeled amount. The proposed method was also applied to monitoring the excretion of cefaclor in human urine samples. It was found that the excretive cefaclor concentration reached its maximum after orally administrated for 1.5 hours, the cefaclor excretive ratio in 8 hours was 51.75%, the total elimination rate constant (Κ) and the half-life time(t1/2) of cefaclor in the body were 0.8063 and 0.86 hours, respectively (in Chapter 5). The interaction of myoglobin with melamine was investigated using luminol-myoglobin CL system. A sensitive chemiluminescence method for the determination of melamine was presented based on the inhibitory effect of melamine on the CL reaction between luminol and myoglobin, and the decrement of CL intensity was proportional to the concentration of melamine ranging from 0.01 to 50.0 ng mL"1 with the detection limit of 3 pg mL-1 (3σ). The proposed method was applied successfully to the determination of melamine in milk products, and the recovery was from 93.4 to 106.5%(in Chapter 6). Based on the inhibitory effect of loratadine on the luminol-myoglobin chemiluminescence reaction, the interaction between myoglobin and loratadine was studied and a CL method for the determination of loratadine was presented. The decrement of CL signal was linear with the logarithm of loratadine concentration over the range from 0.1 to 100.0 ng mL-1 with the detection limit of 0.03 ng mL-1 (3a) and relative standard deviation of less than 3.0%(n= 7). The proposed procedure was applied successfully to obtaining the interaction parameters of myoglobin-loratadine complex and determining loratadine in the medicine of tablets, human serum and urine (in Chapter 7).