English

• In the past decades, the therapeutic potentials of carbon monoxide (CO) have attracted considerable attention^[1-4]. This is because this gaseous molecule possesses cytoprotective, anti-inflammatory, antiproliferative, and anti-apoptotic functions, which are of potential therapeutic applications^[5-7]. To explore the therapeutic potentials of CO, its controllable delivery is highly desired. Since there are several shortcomings in directly administering gaseous CO, CO-releasing molecules (CORMs) have been developed in the past decades aiming at precisely and controllably delivering CO to targeted tissues or organs. Metal carbonyl complexes are classic organometallic compounds of transition metals with CO and release CO upon external stimulations such as solvolysis, ligand exchange, irradiations. Therefore, an increasing number of transition metal compounds have been investigated as CORMs^[8-11]. Among various CORMs, iron carbonyl complexes have attracted considerable attention^[12-18], because not only there are tremendous iron carbonyl complexes available, but also both the complexes themselves and the residues after CO-release are arguably less detrimental compared to other transition metal complexes since iron is one of the essential elements in the human body^[18].

  Of many iron carbonyl complexes, diiron carbonyl complexes have attracted particular attention in the last twenty years or so due to their structural resemblance to the diiron subunit of [FeFe]-hydrogenase and thus, have intensively been investigated as the mimics of the diiron subunit^[19-22]. The high CO-loading per diiron carbonyl complex molecule and capability of releasing CO upon ligand exchange reaction makes them extremely attractive as potential CORMs^{[18, 23-24]}. Our previous reports have demonstrated that these diiron carbonyl complexes could release CO under the stimulation of nucleophilic substitution reactions^[23-24]. A water-soluble diiron hexacarbonyl complex, [Fe₂{ μ-SCH₂CH(OH)CH₂(OH)}₂(CO)₆] (1, Scheme 1), is such a typical example that releases CO with the presence of cysteamine (CysA) or some amino acids^{[23, 25]}.

  Scheme 1

  

  Scheme 1.  Structure of complex 1

  下载: 全尺寸图片 幻灯片

  It is well known that proteins, one category of the essential biological molecules in living organisms, are full of nucleophilic residues. Therefore, the interaction between a CORM and proteins is inevitable. Indeed, the study of such an interaction has been one of the research interests and is of significance in drug design and understanding the pharmacology and biocompatibility of CORMs as drugs. It is known that the concentration of glutathione (GSH) is about 5-fold (ca. 10 mmol·L^-1) higher in cancer cells than in normal cells (ca. 2 mmol·L^-1) ^[26]. Therefore, it is of importance to study the interaction of iron-based CORMs prodrug with GSH since iron could cause Fenton reaction and the released CO can compete with O₂ to bind to cytochrome C in cells. These events in cells can play roles in suppressing cancer growth. In pharmacological investigations, how a drug interacts with biological molecules such as proteins, GSH, DNA is of fundamental importance. Herein, we report our primary study in the interactions of hemoglobin (Hb), myoglobin (Mb), bovine serum albumin (BSA), GSH, and pUC19 plasmid DNA with complex 1 to examine how these biologically relevant molecules promote its CO-release. The kinetics of the CO-release of the complex upon the interaction with these proteins/GSH was examined using IR spectroscopy. Its interactions with these biological molecules were further probed using other spectroscopic techniques, such as UV-Vis, fluorescent, and circular dichroism (CD) spectroscopy. The interactions between the complex and pUC19 were monitored using gel electrophoresis. UV absorption spectra variation and fluorescent quench effect on the proteins in the presence of complex 1 showed interactions between the proteins and complex 1. But CD spectra showed that there was hardly any conformational change in the proteins in the interactions with complex 1. However, the DNA interaction test revealed that the complex could not cause any damage to the DNA.

  1.   Experimental

  1.1   Materials and instrumentations

  All operations were carried out under an argon atmosphere. Mb (equine heart), Hb (bovine), and BSA were purchased from Beijing BioDee Biotechnology Co., Ltd., and GSH, CysA, Na₂HPO₄, NaH₂PO₄ were purchased from Aladdin. Complex 1 was synthesized using the method we reported before^[23]. FTIR spectra were recorded on Agilent 640 using a CaF₂-cell with a spacer of 0.1 mm. UV-Vis spectra were measured on Evolution 201 (Thermo Scientific^TM). Fluorescent spectra were obtained on Varian (Cary Eclipse) with a 10 nm slit for both excitation and emission. Solid-state CD spectra were measured on a CD spectrometer (J-810, Jasco).

  1.2   CO-release monitoring

  A typical procedure for the monitoring was as follows: to a solution of complex 1 (3.3 mg, 0.006 6 mmol) in D₂O (2.0 mL) was added a solution of Mb (50 µL, 4 mmol·L^-1) in phosphate buffer solution (PBS, pH =7.4). The mixture was maintained at 37 ℃ and regularly monitored using IR spectroscopy. The CO-release initiated by Hb (50 µL, 4 mmol·L^-1), BSA (50 µL, 4 mmol·L^-1), GSH (50 µL, 4 mmol·L^-1), and CysA (50 µL, 4 mmol·L^-1), was analogously performed, respectively. The above aqueous solutions were prepared in D₂O since water molecule causes strong interference in a range from 2 000 to 1 900 cm-1. Owing to the limited solubility of complex 1 in D₂O, a minimum DMSO (50 µL) was added to assist the dissolution of complex 1.

  1.3   Collection of UV-Vis and fluorescent spectra

  To facilely compare the interactions of complex 1 with different biological molecules, the concentration of complex 1 and amounts of the biological molecules in the system (3 mL in total) should be kept at the same throughout UV-Vis and fluorescent spectroscopic studies. A typical procedure was as follows: to a solution of Mb (3 mL, 3.3 µmol·L^-1) was added an appropriate amount of complex 1 (33 µmol·L^-1). Then the UV-Vis or fluorescent spectrum of the mixture was recorded. The monitoring for the interaction of complex 1 with the other two proteins was analogously performed.

  1.4   Gel electrophoresis

  The DNA interaction experiments were done using agarose gel electrophoresis at 37 ℃ and a typical procedure was as follows: pUC19 plasmid DNA (0.01 µg·µL^-1) in 50 mmol·L^-1 Tris-HCl+18 mmol·L^-1 NaCl buffer (10 µL, pH=7.2) was treated with complex 1 and CysA. The mixture was incubated for 4 h before loading buffer was added. Then the sample was electrophoresed for 2.5 h at 80 V on 0.8% agarose gel using 20 mmol·L^-1 Tris-boric acid-EDTA buffer. After the electrophoresis, DNA bands were visualized by UV light and photographed.

  2.   Results and discussion

  2.1   CO-release promoted by the proteins

  Hb, BSA, and Mb are common proteins existing in blood or muscle in organisms. GSH is a short-chain peptide composed of three amino acids which is an important antioxidant and radical scavenger in vivo. Therefore, these proteins or peptides were selected in the study to demonstrate how complex 1 interacts with these biologically relevant substances in PBS. In the investigations, the molar ratio of complex 1 over a protein was kept at 10∶1. For comparison, the CO-release initiated by CysA was also carried out under the same conditions. As shown in Fig. 1, the characteristic IR absorption peaks of complex 1 (2 068, 2 032, and 1 989 cm-1) decreased continuously with reaction time in the presence of Hb in PBS. The spectroscopic monitoring results for the CO-release initiated by other substances (Mb, BSA, and GSH) and the stability of complex 1 in PBS are shown in Fig.S1-S4 (Supporting information). For CysA, the spectral variation with reaction time is shown in Fig.S5. For comparison, the CO-release was also examined in the presence of both CysA and BSA (Fig.S6). The results indicate clearly that the presence of these biologically relevant substances can promote CO-release from the diiron carbonyl complex. Since these molecules possess no other capabilities but nucleophilicity, the ligand exchange mechanism for the CO-release initiated by these substances should also apply^[23-25]. The reaction initiated by Hb leads to decompose of complex 1 to completely release CO since no other intermediate bearing CO was detected at the spectroscopic time scale. CysA can react with complex 1 slowly to promote CO-release^[23], and its co-existence with BSA could synergistically accelerate the decomposition of complex 1 (Fig.S6).

  图 1

  

  图 1.  IR spectral variation of complex 1 in the presence of Hb in PBS/D₂O at 37 ℃ under an open atmosphere

  c₁=3.3 mmol·L^-1, c_Hb=0.33 mmol·L^-1

  下载: 全尺寸图片 幻灯片

  Kinetic analysis of the decomposition of complex 1 initiated by an appropriate nucleophilic agent was performed. The linear plots are shown in Fig. 2, which suggest that the reactions between complex 1 and the proteins, GSH or CysA follow a first-order kinetic model. All the kinetic data are summarized in Table 1. For the proteins, Mb showed the most effective interaction in promoting the decomposition of complex 1 whereas the reaction initiated by BSA was the most sluggish one. Compared to the reaction rate constant with BSA, the reaction rates for Mb and Hb were about two and one times faster, respectively. GSH turned out the most effective accelerator to trigger CO release from complex 1 (Table 1). The reason may be attributed to its two carboxylic groups and one thiol group, of which the thiol group is of strong affinity towards iron carbonyl complexes. Further, in PBS (pH=7.4), the carboxylic groups of GSH with an isoelectric point of 5.93 would exist mostly in carboxylate form which would be more nucleophilic. The concentration of GSH was about 5-fold higher than in normal cells. The recent report of our collaborator also revealed that endogenous GSH in cancer cells can trigger the CO release of complex 1 and the releasing rate of CO in cancer cells was faster than that in normal cells^[26]. Therefore, such a CO-releasing system can be of potential as anticancer drugs. Interestingly, both BSA and CysA could jointly accelerate the decomposition of complex 1 demonstrating a synergistic effect (Table 1).

  图 2

  

  图 2.  Plots of the logarithm of IR absorbance of complex 1 vs reaction time in the presence of various nucleophiles in PBS/D₂O

  c₁=3.3 mmol·L^-1, c_nucleophile=0.33 mmol·L^-1; Absorbance at 2 038 cm^-1 was used for the kinetic analysis

  下载: 全尺寸图片 幻灯片

  表 1

  

  表 1  Kinetic data of the decomposition of complex 1 in the presence of different nucleophiles in PBS/D₂O at 37 ℃ under an open atmosphere^a

  下载: 导出CSV

  Nucleophile	kobs/h-1	t1/2/h	pIb
  PBS	(0.8±0.7)x10-2	82.0
  Mb	(9.8±1.1)x10-2	7.1	6.99
  Hb	(6.3±0.1)x10-2	11.0	6.8
  BSA	(3.6±0.9)x10-2	19.3	4.9
  CysA	(0.87±0.5)x10-2	13.0
  BSA+CysAc	(10.5±0.3)x10-2	6.6
  GSH	(11.5±0.1)x10-2	6.0	5.93
  a c1=3.3 mmol·L-1, cnucleophile=0.33 mmol·L-1, t1/2=half time, kobs=apparent rate constant; b pI=isoelectric point; c Combined concentration of BSA and CysA.

  2.2   Spectroscopically probing the interaction between the proteins and complex 1

  UV-Vis spectroscopy is the most adopted method for studying conformation change of proteins. The UV-Vis spectral variations of different proteins and control in PBS (pH=7.4) in the presence of complex 1 are shown in Fig. 3-5 and Fig.S7. The UV-Vis spectrum of complex 1 in PBS showed a strong absorption at 333 nm and a weak shoulder absorption band at 460 nm (Fig. S7), which should be attributed to the electron transition bands of LMCT and d-d transition, respectively. In addition, the UV-Vis absorption of complex 1 decreased steadily with time (Fig. S7), which indicates the slow decomposition of complex 1 in PBS.

  图 3

  

  图 3.  UV-Vis spectra of complex 1 (33 µmol·L^-1) and Hb (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片

  图 4

  

  图 4.  UV-Vis spectra of complex 1 (33 µmol·L^-1) and Mb (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片

  图 5

  

  图 5.  UV-Vis spectra of complex 1 (33 µmol·L^-1) and BSA (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片

  Both Mb and Hb showed two absorption peaks at 280 and 405 nm, which are ascribed to the residue of amino group and Soret peak, respectively (Fig. 3 and 4). BSA just comprised the residue of the amino group and presents one peak at 280 nm (Fig. 5). When complex 1 was gradually added, considerable interactions of complex 1 with these proteins were demonstrated as indicated by the increasing band at 280 nm (Fig. 3-5). However, there was almost no change for the band at 405 nm, the peak belonging to Mb and Hb, which implies that the moiety and coordination sphere of complex 1 is not affected. This is attributed to the protection of the moiety offered by the protein domains. Additionally, a new band at 333 nm appeared and increased gradually, which may be contributed to the absorption band of complex 1. The highly similar spectral change suggests a similar interaction mechanism. These results suggest that the products of complex 1 with these proteins possess a rather similar coordination atmosphere.

  Proteins are always fluorescent owing to the existence of those aromatic amino residues, such as tryptophan, tyrosine, and phenylalanine. As shown in Fig. 6, the fluorescence of the proteins was significantly affected when complex 1 was present. Upon excitation at 280 nm, Hb, Mb, and BSA exhibited considerable fluorescence at 327, 328, and 348 nm, respectively. With the addition of complex 1, the fluorescent intensity of these proteins decreased instantly while band position and spectral profiles did not change. The fluorescent quench effect of the complex on the proteins could be attributed to the interaction of the proteins with complex 1, which agrees with the UV-Vis spectral changes upon the interactions with the proteins as discussed above.

  图 6

  

  图 6.  Fluorescent spectra of different proteins in the absence and presence of complex 1 in PBS

  c₁=33 µmol·L^-1, c_protein=3.3 µmol·L^-1; pH=7.4

  下载: 全尺寸图片 幻灯片

  As discussed above, there are strong interactions between the proteins and the iron carbonyl complex, particularly Hb and Mb. To examine whether the interaction changes significantly the conformation of the proteins, CD spectroscopy was employed to check the reactions and the results are shown in Fig. 7. The CD spectra indicate that the interaction does not lead to significant variations in their conformations. But there were subtle differences among the three proteins. For BSA, the two spectra were nearly superimposable whereas, for both Hb and Mb, there were observable changes in peak intensities. These observations are in good agreement with the interactions of the proteins with the iron carbonyl complex.

  图 7

  

  图 7.  CD spectra of different proteins (a: Hb, b: BSA, c: Mb) in the presence and absence of complex 1

  c₁=30 µmol·L^-1, c_protein=3 µmol·L^-1

  下载: 全尺寸图片 幻灯片

  2.3   Interaction of CORM with DNA

  To examine whether the CO-release and the resultant residues would lead to any DNA degradation, the mixture of CysA and complex 1 was incubated with pUC19 plasmid DNA for 4 h. Analysis of electrophoresis of the DNA suggests no observable DNA degradation during the incubation (Fig. 8).

  图 8

  

  图 8.  Interaction of the CO-releasing system (CysA+1) with pUC19 plasmid DNA

  c₁=0.0, 4.0, 8.0, 16.0, 32.0, 64.0 µmol·L^-1 (from left to right); c_CysA=12.0 µmol·L^-1; ρ_DNA=0.01 µg·µL^-1

  下载: 全尺寸图片 幻灯片

  3.   Conclusions

  In summary, the interactions of a diiron hexacarbonyl complex 1 with Hb, Mb, BSA proteins, and GSH, and their promotion to the CO-release from the complex were studied. UV-Vis and fluorescent spectroscopic investigations suggest that among the examined three proteins, Hb, Mb, and BSA, the interaction of BSA with the complex is the weakest one, which echoes rightly the trend of CO-release promoted by these proteins. All the CO-release follows the first-order kinetic model. In the promotion of CO-release, GSH shows the best efficiency, which may be of significance in exploring anticancer drugs since, in cancer cells, the level of GSH is higher than normal cells. Despite the strong interactions between the proteins and the iron complex, the CO-releasing process and relevant residues do not either significantly alter the conformations of the proteins or degrade pUC19 plasmid DNA.

  Supporting information is available at http://www.wjhxxb.cn

  
  
• 1. [1]

     Babu D, Motterlini R, Lefebvre R A. CO and CO-Releasing Molecules (CO-RMs) in Acute Gastrointestinal Inflammation[J]. Brit. J. Pharmacol, 2015, 172(6):  1557-1573. doi: 10.1111/bph.12632

  2. [2]

     Adach W, Błaszczyk M, Olas B. Carbon Monoxide and Its Donors-Chemical and Biological Properties[J]. Chem. Biol. Interact., 2020, 318:  108973. doi: 10.1016/j.cbi.2020.108973

  3. [3]

     Mann B E. Signaling Molecule Delivery (CO)//Reedijk J, Poeppelmeier K. Comprehensive Inorganic Chemistry Ⅱ. 2nd ed. Amsterdam: Elsevier, 2013: 857-876

  4. [4]

     Kourti M, Jiang W G, Cai J. Aspects of Carbon Monoxide in Form of CO-Releasing Molecules Used in Cancer Treatment: More Light on the Way[J]. Oxid. Med. Cell. Longev., 2017, 2017:  9326454.

  5. [5]

     Motterlini R, Clark J E, Foresti R, Sarathchandra P, Mann B E, Green C J. Carbon Monoxide-Releasing Molecules-Characterization of Biochemical and Vascular Activities[J]. Circ. Res., 2002, 90(2):  E17-E24.

  6. [6]

     Mann B E, Motterlini R. CO and NO in Medicine[J]. Chem. Commun., 2007, (41):  4197-4208. doi: 10.1039/b704873d

  7. [7]

     Mann B E. Carbon Monoxide: An Essential Signaling Molecule[J]. Top. Organomet. Chem., 2010, 32:  247-285.

  8. [8]

     Ford P C. Metal Complex Strategies for Photo-Uncaging the Small Molecule Bioregulators Nitric Oxide and Carbon Monoxide[J]. Coord. Chem. Rev., 2018, 376:  548-564. doi: 10.1016/j.ccr.2018.07.018

  9. [9]

     Ismailova A, Kuter D, Bohle D S, Butler I S. An Overview of the Potential Therapeutic Applications of CO-Releasing Molecules[J]. Bioinorg. Chem. Appl., 2018, :  8547364.

  10. [10]

      Lazarus L S, Benninghoff A D, Berreau L M. Development of Triggerable, Trackable, and Targetable Carbon Monoxide Releasing Molecules[J]. Acc. Chem. Res., 2020, 53(10):  2273-2285. doi: 10.1021/acs.accounts.0c00402

  11. [11]

      Alberto R, Motterlini R. Chemistry and Biological Activities of CO-Releasing Molecules (CORMs) and Transition Metal Complexes[J]. Dalton Trans., 2007, (17):  1651-1660. doi: 10.1039/b701992k

  12. [12]

      Fairlamb I J S, Duhme-Klair A K, Lynam J M, Moulton B E, O'Brien C T, Sawle P, Hammad J, Motterlini R. η⁴-Pyrone Iron(0)carbonyl Complexes as Effective CO-Releasing Molecules (CO-RMs)[J]. Biorg. Med. Chem. Lett., 2006, 16(4):  995-998. doi: 10.1016/j.bmcl.2005.10.085

  13. [13]

      Scapens D, Adams H, Johnson T R, Mann B E, Sawle P, Aqil R, Perrior T, Motterlini R. [(η-C₅H₄R)Fe(CO)₂X], X=Cl, Br, I, NO₃, CO₂Me and[(η-C₅H₄R)Fe(CO)₃]⁺, R= (CH₂)_nCO₂Me (n=0-2), and CO₂CH₂CH₂OH: A New Group of CO-Releasing Molecules[J]. Dalton Trans., 2007, (43):  4962-4973. doi: 10.1039/b704832g

  14. [14]

      Kretschmer R, Gessner G, Görls H, Heinemann S H, Westerhausen M. Dicarbonyl-Bis(cysteamine)iron(Ⅱ) : A Light Induced Carbon Monoxide Releasing Molecule Based on Iron (CORM-S1)[J]. J. Inorg. Biochem., 2011, 105(1):  6-9. doi: 10.1016/j.jinorgbio.2010.10.006

  15. [15]

      Romanski S, Kraus B, Guttentag M, Schlundt W, Rucker H, Adler A, Neudorfl J M, Alberto R, Amslinger S, Schmalz H G. Acyloxybutadiene Tricarbonyl Iron Complexes as Enzyme-Triggered CO-Releasing Molecules (ET-CORMs): A Structure-Activity Relationship Study[J]. Dalton Trans., 2012, 41(45):  13862-13875. doi: 10.1039/c2dt30662j

  16. [16]

      Romanski S, Rücker H, Stamellou E, Guttentag M, Neudörfl J M, Alberto R, Amslinger S, Yard B, Schmalz H G. Iron Dienylphosphate Tricarbonyl Complexes as Water-Soluble Enzyme-Triggered CO-Releasing Molecules (ET-CORMs)[J]. Organometallics, 2012, 31(16):  5800-5809. doi: 10.1021/om300359a

  17. [17]

      Botov S, Stamellou E, Romanski S, Guttentag M, Alberto R, Neudoerfl J M, Yard B, Schmalz H G. Synthesis and Performance of Acyloxy-diene-Fe(CO)₃ Complexes with Variable Chain Lengths as Enzyme-Triggered Carbon Monoxide-Releasing Molecules[J]. Organometallics, 2013, 32(13):  3587-3594. doi: 10.1021/om301233h

  18. [18]

      Jiang X J, Xiao Z Y, Zhong W, Liu X M. Brief Survey of Diiron and Monoiron Carbonyl Complexes and Their Potentials as CO-Releasing Molecules (CORMs)[J]. Coord. Chem. Rev., 2021, 429:  213634. doi: 10.1016/j.ccr.2020.213634

  19. [19]

      Liu X M, Ibrahim S K, Tard C, Pickett C J. Iron-Only Hydrogenase: Synthetic, Structural and Reactivity Studies of Model Compounds[J]. Coord. Chem. Rev., 2005, 249(15/16):  1641-1652.

  20. [20]

      Tard C, Liu X M, Ibrahim S K, Bruschi M, De Gioia L, Davies S C, Yang X, Wang L S, Sawers G, Pickett C J. Synthesis of the H-Cluster Framework of Iron-Only Hydrogenase[J]. Nature, 2005, 433(7026):  610-613. doi: 10.1038/nature03298

  21. [21]

      Tard C, Pickett C J. Structural and Functional Analogues of the Active Sites of the[Fe]-, [NiFe]-, and[FeFe]-Hydrogenases[J]. Chem. Rev., 2009, 109(6):  2245-2274. doi: 10.1021/cr800542q

  22. [22]

      Li Y L, Rauchfuss T B. Synthesis of Diiron(Ⅰ) Dithiolato Carbonyl Complexes[J]. Chem. Rev., 2016, 116(12):  7043-7077. doi: 10.1021/acs.chemrev.5b00669

  23. [23]

      Long L, Jiang X J, Wang X, Xiao Z Y, Liu X M. Water-Soluble Diiron Hexacarbonyl Complex as a CO-RM: Controllable CO-Releasing, Releasing Mechanism and Biocompatibility[J]. Dalton Trans., 2013, 42:  15663-15669. doi: 10.1039/c3dt51281a

  24. [24]

      Jiang X J, Long L, Wang H L, Chen L M, Liu X M. Diiron Hexacarbonyl Complexes as Potential CO-RMs: CO-Releasing Initiated by a Substitution Reaction with Cysteamine and Structural Correlation to the Bridging Linkage[J]. Dalton Trans., 2014, 43(26):  9968-9975. doi: 10.1039/C3DT53620C

  25. [25]

      Chen L M, Jiang X J, Wang X, Long L, Zhang J Y, Liu X M. A Kinetic Analysis of CO Release from a Diiron Hexacarbonyl Complex Promoted by Amino Acids[J]. New J. Chem., 2014, 38(12):  5957-5963. doi: 10.1039/C4NJ00661E

  26. [26]

      Gao C J, Liang X H, Guo Z X, Jiang B P, Liu X M, Shen X C. Diiron Hexacarbonyl Complex Induces Site-Specific Release of Carbon Monoxide in Cancer Cells Triggered by Endogenous Glutathione[J]. ACS Omega, 2018, 3(3):  2683-2689. doi: 10.1021/acsomega.8b00052

• 

  Scheme 1  Structure of complex 1

  下载: 全尺寸图片 幻灯片
  

  图 1  IR spectral variation of complex 1 in the presence of Hb in PBS/D₂O at 37 ℃ under an open atmosphere

  c₁=3.3 mmol·L^-1, c_Hb=0.33 mmol·L^-1

  下载: 全尺寸图片 幻灯片
  

  图 2  Plots of the logarithm of IR absorbance of complex 1 vs reaction time in the presence of various nucleophiles in PBS/D₂O

  c₁=3.3 mmol·L^-1, c_nucleophile=0.33 mmol·L^-1; Absorbance at 2 038 cm^-1 was used for the kinetic analysis

  下载: 全尺寸图片 幻灯片
  

  图 3  UV-Vis spectra of complex 1 (33 µmol·L^-1) and Hb (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片
  

  图 4  UV-Vis spectra of complex 1 (33 µmol·L^-1) and Mb (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片
  

  图 5  UV-Vis spectra of complex 1 (33 µmol·L^-1) and BSA (3.3 µmol·L^-1) in PBS (pH=7.4) in the presence of complex 1 with various concentrations

  下载: 全尺寸图片 幻灯片
  

  图 6  Fluorescent spectra of different proteins in the absence and presence of complex 1 in PBS

  c₁=33 µmol·L^-1, c_protein=3.3 µmol·L^-1; pH=7.4

  下载: 全尺寸图片 幻灯片
  

  图 7  CD spectra of different proteins (a: Hb, b: BSA, c: Mb) in the presence and absence of complex 1

  c₁=30 µmol·L^-1, c_protein=3 µmol·L^-1

  下载: 全尺寸图片 幻灯片
  

  图 8  Interaction of the CO-releasing system (CysA+1) with pUC19 plasmid DNA

  c₁=0.0, 4.0, 8.0, 16.0, 32.0, 64.0 µmol·L^-1 (from left to right); c_CysA=12.0 µmol·L^-1; ρ_DNA=0.01 µg·µL^-1

  下载: 全尺寸图片 幻灯片

  表 1  Kinetic data of the decomposition of complex 1 in the presence of different nucleophiles in PBS/D₂O at 37 ℃ under an open atmosphere^a

  Nucleophile	kobs/h-1	t1/2/h	pIb
  PBS	(0.8±0.7)x10-2	82.0
  Mb	(9.8±1.1)x10-2	7.1	6.99
  Hb	(6.3±0.1)x10-2	11.0	6.8
  BSA	(3.6±0.9)x10-2	19.3	4.9
  CysA	(0.87±0.5)x10-2	13.0
  BSA+CysAc	(10.5±0.3)x10-2	6.6
  GSH	(11.5±0.1)x10-2	6.0	5.93
  a c1=3.3 mmol·L-1, cnucleophile=0.33 mmol·L-1, t1/2=half time, kobs=apparent rate constant; b pI=isoelectric point; c Combined concentration of BSA and CysA.

  下载: 导出CSV
•