Study On Phenoloxidase-like Properties And Antibacterial Activity Of Hemoglobin From Blood Clam Scapharca Kagoshimensis

Blood clam (Scapharca kagoshimensis) is a genus of commercial valuable marine invertebrate belongs to Mollusca, Lamellibranchia. Presence of hemoglobin (Hb) in blood cells makes it an exception in bivalves. Lacking of T-cell, true immune antibody and specific immune system like vertebrates, the defense to external pathogen invasion in blood clam relies on its innate immune response exerted by blood cells and humoral fluid factors, in which blood cells play a pivotal role. Hb is a class of ferritin with reversible oxygen-carrying ability existing in all vertebrates and some invertebrates. It was discovered recently that Hb contributed also to blood pressure maintaining, bacterial inhibition, sulfide transportation, pH balance adjustment etc. in addition to its duty as an oxygen carrier. Thus it is highly valued in biological research. It is proposed in this project to isolate and purify two types of Hb, Hbâ… and Hbâ…¡in the configuration of dimer and tetramer from blood cell lysate of blood clam, to characterize its biochemical properties and verify its phenoloxidase (PO)-like activity, to explore its antibacterial activity in vitro using Staphylococcus aureus, Bacillus subtilis, Micrococcus, Eschetichia coli, Clostridium perfringens, Proteus species, Vibrio anguillarum, Vibrio harveyi, Penicillium glaucum, Aspergillus niger, Saccharomyces cerevisiae and Schizosaccharomyces pombe as indicators, to investigate its antibacterial mechanism using the combination of 2-Methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo[1, 2-a] pyrazin-3-one (MCLA) as a chemiluminescent probe and specific inhibitory effect of SOD against ROS. It is targeted to reveal functions of Hb in non-specific immune defense and lay a foundation for further discovery in its molecular mechanism.In this study, three Hb fractions were purified from blood cell lysate of blood clam using sephacryl S-100 gel-filtration and Amicon Ultra-4 centrifugal filtration device, presenting three bands at 30.28 kDa, 28.9 kDa and 31.2 kDa on SDS-PAGE, respectively. Fractions in two lower bands were identified as the two subunits of Hbâ…¡while protein presenting the 31.2 kDa band was identified as Hbâ… using mass spectrometry. Molecular weight of Hbâ…¡obtained from gel filtration analysis was 57.8 kDa, which is consistent with data stated above. It is suggested by enzymatic activity assay results that both Hbâ…¡and Hbâ… exhibit PO-like activity against L-DOPA as a substrate in non-activating natural condition. Their PO-like activity can be further activated by isopropanol but cannot be activated by SDS, trypsin or urea.Biochemical and enzymatic characterization results indicated that optimal temperature and pH for PO-like activity presented by blood clam Hbâ…¡against L-DOPA were 40°C and 7.0 while these parameters of Hbâ… were 50°C and 7.0, respectively. Neither Hbâ…¡nor Hbâ… can oxidise tyrosine or phenol although they both displayed PO-like activity against L-DOPA, catechol and hydroquinone. Hbâ…¡'s Km against L-DOPA, catechol and hydroquinone were 1.22 mM, 2.71 mM and 0.97 mM while that of Hbâ… were 2.0 mM, 5.27 mM and 1.55 mM under optimal conditions, suggesting both Hbâ…¡and Hbâ… are PO-like enzyme with catecholase and polyphenol oxidase properties. Inhibition results showed that sodium sulfite, ascorbic acid, citric acid and cysteine suppressed PO-like activity from Hbâ…¡and Hbâ… strongly while benzoic acid, phenylthiocarbamide and thiourea performed comparative weaker inhibitory effects, suggesting blood clam Hbâ…¡and Hbâ… are more similar to catecholase when exhibiting PO-like activity. Results for metal chelators and cations analysis indicated that EDTA inhibited the PO-like activity from Hbâ…¡and Hbâ… seriously while DETC, Cu²⁺, Zn²⁺ , Mg²⁺ and Ca²⁺ inhibited them to different extents. However, Fe²⁺ performed a significant activating effect on their PO-like activity and could completely restore their activity inhibited by EDTA. It is therefore implied that blood clam Hbâ…¡and Hbâ… are both metalloenzyme, which is consistent with their property as ferritins. Conclusively, blood clam Hbâ…¡and Hbâ… present dual properties of catecholase and polyphenol oxidase at the same time but more similar to a Fe²⁺-containing catecholase when exhibiting PO-like activity.Data from blood clam Hb antibacterial activity in vitro indicated that MIC of blood clam Hbâ…¡towards three types of gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis and Micrococcus tetragenus were 0.373 mg/mL, 0.186 mg/mLand 0.047 mg/mL while corresponding data of Hbâ… were 1.910 mg/mL, 0.060 mg/mL and 0.060 mg/mL. However, neither of them showed antibacterial activity towards some types of gram-negative bacteria including Eschetichia coli, Clostridium perfringens and Proteus species, Vibrio anguillarum, Vibrio harveyi and fungi including Penicillium glaucum, Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomyces pombe. Blood clam Hb antibacterial mechanism exploration using MCLA as a chemiluminescent probe indicated that Hbâ…¡and Hbâ… could generate ROS which were specific inhibited by SOD, suggesting ROS generated by Hbâ…¡and Hbâ… were superoxide anions. It can be concluded from above data that blood clam Hbâ…¡and Hbâ… present antibacterial activity against gram-positive bacteria by generating superoxide anion ROS.This thesis is the first report on PO-like activity from blood clam Hb and its non-specific immune activity. It is therefore important for the elucidation of the involvement of Hb in invertebrate non-specific immune system as well as the multi-biological functions of respiratory proteins.