Biol Trace Elem Res (2009) 127:269–277 DOI 10.1007/s12011-008-8243-3 Study of Antibacterial Activity of ZnZnbisporphyrin Complexes and its Free Components on Staphylococcus aureus by Microcalorimetry Xiang-jiao Xu & Chun-qiao Chen & Zhuo-yuan Wang & Yi Zhang & An-xin Hou & Chao-Hong Li & Yi Liu Received: August 2008 / Accepted: 16 September 2008 / Published online: November 2008 # Humana Press Inc 2008 Abstract The antibacterial effect of Zn(II), tetraphenyl porphyrin (TPP), propdioxyl bridged tetraphenyl bisporphyrin 1, and its metallobisporphyrin complexes (ZnMnbisporphyrin and ZnZnbisporphyrin 3) towards Staphylococcus aureus growth was investigated by microcalorimetry at 37°C Differences in their capacities to inhibit the growth metabolism of S aureus were observed By analyzing the power–time curves, crucial parameters such as the rate constant of bacterial growth (k), inhibitory ratio (I), and generation time (tG) were determined The growth rate constant (k) of S aureus (in the log phase) in the presence of the drugs decreased linearly with increasing concentrations of the complexes The sequence of the antibacterial activities of these compounds tested was 3>2>1>Zn(II)> TPP ZnZnbisporphyrin is proposed to benefit from the synergetic effects of Zn(II) and Keywords Bisporphyrin complexes Staphylococcus aureus Antibacterial effects Microcalorimetry Thermokinetics Introduction Multiple resistance to antibiotics is a growing concern in hospitals due to the difficulty in eradicating them [1–2] In order to overcome these resistance problems, there is an urgent need for discovering novel antibacterial agents belonging to structural classes distinct from existing antibiotics [3–6] Porphyrins have received considerable interest due to their ability X.-j Xu : C.-q Chen : Z.-y Wang : Y Zhang : A.-x Hou : Y Liu (*) State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China e-mail: prof.liuyi@263.net X.-j Xu College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan, Hubei 430081, China C.-H Li School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430072, China 270 Xu et al to act as photosensitizers when irradiated with visible light [7–11] Because porphyrin analogous can take cytotoxic singlet oxygen directly in tumor cells causing cell death when irradiate, photosensitization can represent a useful approach to kill microbial In an effort to identify new antibacterial compounds and in connection with our research programe on porphyrins, we synthesized propdioxyl bridged tetraphenyl bisporphyrin and its metallobisporphyrin complexes (ZnMnbisporphyrin and ZnZnbisporphyrin 3) Free base bisporphyrins are suitable ligands that are able to coordinate two metal ions This feature renders them good candidates for synthesis of dinuclear complexes, which might exert stronger antibacterial activity than monoporphyrin complexes [12–16] Zinc is an element that is essential for cell proliferation and differentiation It is recognized as one of the most important biological cofactors relatively late because it does not absorb light like iron and copper Zinc is also a structural constituent of a great number of proteins including metabolic enzymes, cellular signaling proteins, and transcription factors [17] Systematic study, herein, of the biological effect of zinc(II), tetraphenyl porphyrin (TPP), bisporphyrin complexes (1, 2, and 3) may, thus, reveal interesting structure–activity relationships (SAR) Microcalorimetry as a technique has been widely used in life sciences because of its high sensitivity, high accuracy, and automaticity [18] Microcalorimetry can provide a continuous measurement of heat production and supply the power–time curves, which can describe the growth process without disturbing the normal activity of the biosystem [19–21] Therefore, it has been extensively used to study the interactions between drugs and cells based on the thermodynamic and kinetic information [22–24] In this paper, the microcalorimetric technique has been used to investigate the effects of Zn(II), TPP, and bisporphyrin complexes (1, 2, and 3) towards Staphylococcus aureus growth and the relationship between the rate of heat production and microbial growth The results are very important for clinic applications of the antibiotics Materials, Instruments, and Methods Materials Bisporphyrin complexes (1, 2, and 3) and TPP were synthesized and characterized by the Department of Chemistry, Wuhan University, P R China [25] The structure of them is shown in Fig Zinc acetate [Zn(CH3COO)2·2H2O) was obtained from Chengdu Chemical corporation All other chemicals used are of analytical grade and available locally S aureus Culture S aureus (CCTCC AB910393) was provided by China Center for Type Culture Collection, Wuhan University, P R China Briefly the broth culture medium in a total volume of 1,000 ml contained NaCl (5 g), beef extract (6 g), peptone (10 g), and pH 7.0 The volume of the container is 100 ml, and the volume of the culture medium was 25 ml The culture medium was sterilized in high-pressure steam at 121–126°C for 30 S aureus were inoculated in 25 ml peptone culture medium and incubated in the shaker for 10 h at 37°C These cells were prepared for microcalorimetric measurements The rotate speed of incubator shaker is 100 rpm The flask is enveloped with cotton plug, so there is enough oxygen, which can be used by S aureus Antibacterial Activity of ZnZnbisporphyrin Complexes N 271 N M N N TPP: M=2H N N N N N M1 N M1 M2 N M2 N O(CH2)3O 2H 2H Mn Zn Zn Zn Fig Structures of TPP and bisporphyrin complexes (1, 2, and 3) LB Medium The bacteria were grown at pH 7.0–7.2 on a Luria–Bertain (LB) prepared from NaCl (5 g), peptone (10 g), and yeast extract (5 g) The medium was sterilized by autoclaving at 0.1 MPa and 120°C for 30 and stored in a refrigerator Instruments A heat-conduction microcalorimeter, LKB-2277 Bioactivity Monitor manufactured by LKB corporation of Sweden, was used to obtain the metabolic growth power–time curves of the bacteria The microcalorimeter was thermostated at 37°C The voltage signal was recorded by means of an LKB-2210 recorder (1,000 mV range) The baseline stability was 0.2 µW/ 24 h For details of the performance and structure of the instrument, see reference [26] Methods In the calorimetric experiment, the flow cell was completely cleaned and sterilized The procedure was as follows: sterilized distilled water, NaOH (0.1 mol/l), alcohol solution (75%), HCl (0.1 mol/l), and sterilized distilled water were pumped in sequence by an LKB2132 microperplex peristaltic pump at a flow rate of 50 ml/h Once the system was cleaned and sterilized and the baseline had been stabilized, the bacterial suspension, initially containing 2×106 bacteria/ml and the appropriate compound [Zn(II), TPP, 1, or 3], was pumped through the calorimetric cell with an LKB-2132 perplex peristaltic pump at a flow rate of 50 ml/h When the flow cell (volume, 0.6 ml) was filled, the pump was stopped and the monitor was used to record the power–time curves of the bacterial growth [18] In this type of experiment, the appropriate compound [Zn(II), TPP, 1, or 3] was added from the beginning of the experiment; they were introduced as soon as the bacteria were 272 Xu et al inoculated in the peptone culture medium All calorimetric experiments were conducted at 37°C Results The Power–Time Curves of S aureus Figure shows the power–time curves obtained when a culture of the test bacteria was inoculated with the appropriate compounds [Zn(II), TPP, 1, or 3] at different con- 20µW 20µW Zn (µmol/L) 200 400 600 t (min) 800 TPP [µg/mL] 326 652 978 1304 1630 P(µW) P(µW) 15.4 46.2 77 308 616 770 924 1232 1000 200 400 20µW 20µW 500 t (min) 1000 1500 1000 10 30 50 100 150 250 300 P(µW) P(µW) 800 (µmol/ L) (µmol/ L) 10 30 50 100 150 200 230 300 600 t (min) 500 t (min) 1000 1500 20µW (µmol/ L) P(µW) 10 30 50 100 150 200 230 300 500 1000 1500 2000 t (min) Fig The power–time curves of S aureus growth in the presence of the appropriate compound [Zn(II), TPP, 1, 2, or 3] at different concentrations Antibacterial Activity of ZnZnbisporphyrin Complexes 273 centrations It can be seen that the shape of the metabolic thermogenic curves changed little when the tested compounds at low concentrations were added into the suspension of the bacterium But when the tested compounds at high concentrations were added, the shapes changed obviously and the lag phase, which was between the start of the experiment and the ascending phase of the power–time curve, became longer with increasing of concentration These curves show that the tested compounds had inhibiting action on the bacterial growth Growth Rate Constant of S aureus and Inhibition Ratio The growth power–time curves of S aureus show that the log phase of growth obeys the equation [18]: ln Pt ẳ ln P0 ỵ kt 1ị Here, P0 and Pt are the heat output power at time and t, respectively Using this equation, the growth rate constant (k) of all the experiments is calculated by analyzing the date of the first peak Furthermore, the generation time (tG), an important parameter in microbiology, can be derived from the equation [18]: tG ẳ ln 2=kc 2ị Here, kc represents the rate constant of the growth of S aureus in the presence of a given compound at concentration c In our experiments, tG of the control agreed well with the literature value [27] The corresponding values between k and tG are shown in Table The growth inhibition ratio is calculated on the basis of the growth rate constant, and inhibitory ratio can be defined as [18]: I ẳ ẵk0 kc Þ=k0 Â 100% ð3Þ Here, k0 is the growth rate constant of the control for S aureus The values of I are also shown in Table Relationship Between the Growth Rate Constant and Concentration To further compare the effect of Zn(II), TPP, 1, 2, and 3, the relationship between the growth rate constant (k) and concentration (c) was studied As can been seen from Table 1, an increase in compound concentration c caused a decrease in growth rate k of S aureus As for Zn(II), k at low concentrations was higher than or close to that of the control This indicates that, at low concentration, Zn(II) stimulates the growth of S aureus For antibacterial assay, we thus focused on concentrations at which the growth rate was less than that of the control Thus, the relationship between k and c were described in Table The above experimental relationships show how the growth rate constant (k) changed as a function of the concentration of the compounds Thus, the growth rate constant (k) decreased most rapidly for This result demonstrates that ZnZnbisporphyrin benefits in a synergetic manner from the inherent properties of its free components Zn(II) and Relationship Between the Inhibition Ratio (I) and Concentration (c) Usually, the inhibition ratio I increased with the increase of the appropriate compound [Mn(II), TPP, or 2] concentration, this can be seen from the date of Table The variation 274 Xu et al Table Thermokinetic Data for the Growth of S aureus with Different Drugs at 37°C Drug c (µmol/l) k (min−1) Ra Zn(II) 15.4 46.2 77 308 616 770 924 1,540 326 652 978 1,304 1,630 10 30 50 100 150 250 300 10 30 50 100 150 200 230 300 10 30 50 100 150 200 230 300 0.02673 0.02692 0.02598 0.02507 0.02304 0.01888 0.01693 0.01377 0.0000 0.03099 0.02850 0.02617 0.02545 0.02484 0.02321 0.03295 0.03108 0.02929 0.02702 0.02607 0.02466 0.02132 0.01977 0.03128 0.02934 0.02852 0.02678 0.02504 0.02351 0.02215 0.02134 0.01763 0.03528 0.03022 0.02880 0.02690 0.02559 0.02465 0.02271 0.01917 0.01650 0.9972 0.9983 0.9966 0.9923 0.9988 0.9973 0.9875 0.9906 0.9904 0.9992 0.9993 0.9990 0.9956 0.9984 0.9987 0.9977 0.9975 0.9982 0.9961 0.9995 0.9985 0.9980 0.9988 0.9972 0.9964 0.9992 0.9982 0.9990 0.9978 0.9984 0.9952 0.9943 0.9955 0.9974 0.9985 0.9995 0.9986 0.9989 0.9980 0.9985 0.9993 TPP a tG (min) 25.93 25.75 26.68 27.65 30.08 36.71 40.94 50.34 22.37 24.32 26.49 27.24 27.90 29.86 21.04 22.30 23.66 25.65 26.59 28.11 32.51 35.06 22.16 23.62 24.30 25.88 27.68 29.48 31.29 32.48 39.32 19.65 22.94 24.07 25.77 27.09 28.12 30.52 36.16 42.01 I (%) 0.00 -0.71 2.81 6.21 13.80 29.37 36.66 48.48 100.00 0.00 8.03 15.55 17.88 19.85 25.10 0.00 5.68 11.11 18.00 20.88 25.16 35.30 40.00 0.00 6.20 8.82 14.39 19.95 24.84 29.19 31.78 43.64 0.00 14.34 18.37 23.75 27.47 30.13 35.63 45.66 53.23 Correlation coefficient tendency of inhibition ratio with concentration was varied with different complexes, which suggested that the mode of action, drug absorption, etc were different from different drugs Figure showed the relationship between I and c From the Fig 3, it can be indirectly seen that the inhibition ratio I increased most rapidly for Thus, the observation of the relationship between I and c further confirms that benefits from a synergetic effect in terms of its inhibitory action toward S aureus Antibacterial Activity of ZnZnbisporphyrin Complexes 275 Table The Relationship Between k and c with Different Drugs at 37°C Drug k–c Zn(II) TPP k=0.02785–1.666×10−5 k=0.03014–7.229×10−6 k=0.02885–2.988×10−5 k=0.02931–3.752×10−5 k=0.03023–4.421×10−5 a Ra c c c c c −0.9617 −0.9239 −0.9967 −0.9920 −0.9824 (15.4–1,540 µmol/l) (0–1,630 µmol/l) (50–300 µmol/l) (10–300 µmol/L) (10–300 µmol/l) Correlation coefficient Discussion Analyses of the power–time curves of S aureus under the action of five kinds of compounds [Zn(II), TPP, 1, or 3] showed that with increasing concentrations of these compounds, the lag phase became longer and the generation time (tG) delayed All of these phenomena suggest that the five compounds all have the capacity to inhibit the growth a Zn(II) TPP 80 I (%) 60 40 20 0 400 800 1200 1600 c (µmol/L) b 60 I = 14.31+ 0.1253c, R=0.9824(10-300 µmol/ L) 50 40 I(%) Fig Relationship between I and c for a the appropriate compound [Zn(II), TPP, 1, 2, or 3], concentration ranges: [Zn(II)]=0–1,232 µmol/l; [TPP]=0–1,630 µmol/L; [1]=0– 300 µmol/l; [2]=0–300 µmol/l; [3]=0–300 µmol/l b ZnZnbisporphyrin 3, concentration ranges: [3]=10– 300 µmol/l 30 20 10 50 100 150 c (µmol/L) 200 250 300 276 Xu et al metabolism of S aureus to different extents and the inhibitory extent varied with the different drugs In the case of Zn(II), the relationships of k and I vs c indicate that Zn(II) has a stimulating effect on the growth of S aureus at low concentration but an inhibitory one at high concentration The biphasic function was interpreted as follows: At the lowest concentration tested, Zn(II) is taken up by the cells and protects their structural integrity It also appears to improve the flow characteristics of the cells and to help maintain their growth by stimulating the intracellular biosynthesis of DNA and RNA While at the high concentration, Zn(II) has growth inhibitory It might be explained that Zn(II) acts on the mercapto of the biological macromolecule and makes the proteins wrecked and the proliferation is rejected The growth rate of the cells decreases In the case of TPP and 1, the structure of cell membrane is most likely altered as a result of the affinity of porphyrin to biological organisms Thus, an enhanced activity is observed for structure bearing two porphyrin groups Finally, the synergetic function of and on S aureus could be due to the unique structure of two complexes carrying simultaneously Zn(II)–Zn(II) di-nuclear or Zn(II)–Mn (II) di-nuclear unit and bisporphyrin group, the porphyrin ring being compatible with the biological structure But considering the rate constant, it could be concluded that gave the better inhibitory effect on S aureus than 2, as Fig shows The action of the drugs on the bacteria also depended on the structure of the drugs Because ionic radius of manganese is larger than that of zinc, it is more difficult to enter into the bacterial cell Thus the inhibition of on the bacteria is smaller than that of All these factors rationalize why has a higher affinity to the bacterial cell membrane than Zn(II), 1, and alone To better understand this phenomenon from a mechanistic point, further studies are required, though Conclusion In conclusion, microcalorimetry is a powerful technique for monitoring and controlling the growth process of microbes It provides kinetic and thermodynamic information that cannot be obtained by conventional bacteriological techniques Furthermore, all of these information are very significant for the synthesis of antibiotics and the studies of toxicology and pharmacology The result shows that these compounds are potential anti-bacteria reagent, and their inhibitory capacities are concentration-dependent The sequence of antibiotic activity of these compounds was 3>2>1>Zn(II)>TPP ZnZnbisporphyrin is proposed to benefit from the synergetic effects of Zn(II) and Acknowledgments We gratefully acknowledge the financial support of project supported by the National Natural Science 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Fig The power–time curves of S aureus growth in the presence of the appropriate compound [Zn(II), TPP, 1, 2, or 3] at different concentrations Antibacterial Activity of ZnZnbisporphyrin Complexes... observation of the relationship between I and c further confirms that benefits from a synergetic effect in terms of its inhibitory action toward S aureus Antibacterial Activity of ZnZnbisporphyrin... Res 36:697–705 Chopra I (1998) Research and development of antibacterial agents Curr Opin Microbiol 1:495–501 Antibacterial Activity of ZnZnbisporphyrin Complexes 277 Spellberg B, Powers JH, Brass