tronger as compared to the ferric forms [15], and is even stronger in COHb as compared to oxy form [9]. Thus it was important to find out if CORM-2 can compete with ferrous heme. Addition of 1000 µM CORM-2 to oxyHb, the concentration at which a fast reaction with met form observed, resulted in an increased absorption and a significant red shift of the Soret peak within mixing time (Figure 2). The latter alterations point at transformation of oxyHb (414 nm peak) to COHb (419 nm peak). However, while the theoretically, the complete transformation should yield a 1.52-fold increase in OD [9], only a 1.16-fold increase was observed (Figure 2). Moreover, following 2 hours, the absorption of the mixture further decreased while the Soret features remained typical for COHb. These data indicate that despite the carboxy-complex formation, a state which strengthens the heme-globin interaction, CORM-2 could still compete with the heme on the site [9].

Identical experiments were performed using oxyMb. Addition of 1000 µM CORM-2 caused a slight red shift

Figure 2. Effect of CORM-2 on Soret spectra of oxyRH. Lines 1 (thin): oxyhb/Mb alone; Lines 2 (gray): Hb/Mb+ CORM-2 (1000 µM) at mixing time; Lines 3 (heavy): 2, following 2 hours of incubation in RT. RH concentration −10 µM.

within the mixing time, indicating only negligible transformation of oxyMb into its carboxy form, although over time the Soret absorption decreased (Figure 2). The difference between Hb and Mb appears to be related to the fact that affinity of CO for Mb (FeII) is not as high and thus conversion of oxy to carboxy form is less efficient than in the case of Hb [9].

To evaluate whether the interaction of CORM-2/ “i”CORM with hydrophobic sites in proteins is a more general phenomenon, similar studies were performed with plasma proteins known to contain amphipathic sites for heme and/or other small molecules.

3.3. Effect of CORM-2 and “i”CORM on Plasma Heme Binding Proteins

Hemopexin (Hx) has a uniquely high affinity (>1013) for the ferric heme. Its single specific heme binding site is composed of eight hydrophobic residues in addition to coordination bonds [16]. Formation of the heme-hemopexin complex (H-Hx) results in an immediate appearance of the typical 414 nm Soret peak (Figure 3 (upper), spectrum 2), which known not to be affected by CO gas [12]. Addition of 1000 µM CORM-2/“i”CORM to H-Hx did not affect the Soret absorption band. Nonetheless, preincubation of CORM-2/“i”CORM with apoHx followed by heme addition resulted in formation of a much smaller Soret absorption (Figure 3 (upper), spectrum 3).

That H-Hx was not affected by CORM-2/“i”CORM can be explained by the fact that heme binding to apoHx is followed by global conformational changes, shielding the heme from the water phase [17]. In the case of heme interaction with the CORM-2/“i”CORM preincubated apoHx, the residual Soret peak at 414 nm is interpreted as competitive binding of the above compounds to the Hx heme binding site. An additional and/or alternative explanation is the possibility that CORM-2/“i”CORM binding, like heme, is followed by protein conformational changes resulting in inaccessibility of the site to any ligand.

Figure 3. Effect of “i”CORM-2 on hemin binding to plasma proteins. Upper: Hx (12.5 µM, by activity—see methods); Lower: HSA (12 µM). Lines 1: “i”CORM alone (1000 µM per dimer); Lines 2: Typical H-Hx/H-HSA complex formed by 10 µM of heme in PBS containing no “i”CORM; Lines 3: A mixture of hemin with apoHx/HSA pre-incubated for 5 minutes in presence of “i”CORM (1000 µM per dimmer).

It was of interest to determine if CORM-2/“i”CORM can interact with human serum albumin (HSA), known to have a single high affinity heme binding site as well as several non-specific amphipathic sites [18,19]. Experiments similar to those carried out with Hx were repeated employing HSA, using hemin concentration allowing only its high affinity site to be potentially occupied. In this case as well, addition of CORM-2/“i”CORM did not affect the spectrum of the preformed heme-albumin complex (H-HSA). Addition of heme following pre-incubation of HSA with CORM-2/“i”CORM resulted in similar Soret spectrum but with decreased OD (Figure 3 (lower), spectrum 3).

Thus, heme binding capacity of both Hx and HSA was lost by CORM-2/“i”CORM treatment but the leftover heme binding sites were unchanged as identified by Soret. To estimate the fraction of remaining heme sites, ΔOD of the Soret peak (414 nm for Hx and 404 nm for HSA) and isosbestic wavelength at the red part of the spectrum (where no contributions from the Soret band are available) was calculated. These calculations indicated that 62% of Hx and 37% of HSA still bound heme in presence of CORMs.

That in comparison to Hx, albumin lost less of its heme binding capacity, despite weaker heme affinity, can be explained by CORMs hydrophobicity. CORMs appears to be less available for the high affinity heme binding site as it is mostly bound to the broad range of non specific sites of HSA, while in Hx it only competes for the single heme-site.

Since CORMs are designed to be applicable for clinical use it should be noted that binding of CORM/ “i”CORM to Hx may prevent its vital physiological activeties such as clearance of differentiating erythroid progenitors membrane from accumulated hemin [20]. In the case of albumin, the major blood protein which binds non specifically low molecular weight amphipathic drugs [21], administration of CORM might alter their pharmacokinetics.

3.4. CORM-2 Effects on Cell Membranes

The hydrophobic nature of CORM-2/“i”CORM binding implies possible general nonspecific association with hydrophobic surfaces of proteins as well as cell membranes. Thus we further analyzed CORM-2/“i”CORM effect on whole blood.

CORM-2/“i”CORM (1000 µM) or DMSO alone as control were added to fresh whole blood samples (see methods). Representative results are demonstrated in Figure 4(a) micrographs. As can be seen, aggregates composed of few red cells were formed by addition of CORM-2/“i”CORM but were not observed in presence of DMSO alone. Previous literature reported red blood cells (RBC) aggregation in blood smears from patients that have received hydrophobic drugs [22]. Thus the aggregates formation in the presence of CORM-2/“i” CORM is consistent with their hydrophobic nature.

To shed further light on the RBC membrane changes which might have caused aggregation, diluted isolated RBCs which yield the same CORM/Hb ratio as in the first section of the study were incubated with 1000 µM CORM-2. Large brown non-resuspendable clumps of precipitate immediately formed leaving no soluble free Hb in the buffer. Therefore we lowered the concentration of CORM-2 to 100 µM which resulted in time dependent hemolysis. Quantitation of the hemolysis was estimated by cell-free oxyHb concentration following 6 hours of incubation (as oxidation/denaturation developed later). The results are shown in Figure 4(b). As can be seen, DMSO alone produced 13% hemolysis while 100 µM of CORM-2 produced a significant increased hemolysis. These results could not be attributed to stabilizing the ferrous heme-iron by the CO liberated from the donor molecule [15]. The hemolysis can be explained by the hydrophobic nature of CORM-2 and all of its intermediates during CO release, allowing their intercalation within

Figure 4. Effect of CORM-2/“i”CORM on red cell membranes. (a) CORM-2/“i”CORM induced red cells aggregation in whole blood. Upper: fresh blood + DMSO 0.5%; Middle: As upper plus CORM-2 (1000 µM); Lower: As upper plus “i”CORM (1000 µM per dimer). (2 µl of blood exposed to the reagent was mixed with 1.5 ml PBS and placed in hemacytometer for microscopic observation.) (b) CORM-2 induced hemolysis in RBC suspension. Acellular oxyHb relative concentrations in buffer following RBC suspension incubation (t = 6 h) in presence of CORM-2 (100 µM) or DMSO alone (control) presented as a percent of maximal value reached by hypotonic hemolysis. Data from 2 donors and 3 repetitions in each.

cell membrane and its deterioration, a general phenomenon of hydrophobic detergents.

Finally, we have shown in the above study that CORM-2 provides non-specific, hydrophobic interactions with a variety of amphipathic surfaces. Such effects appear to be unrelated to CO activity. This observation indicates that during the gas release the intermediate states of hydrophobic complementary core molecules afford this activity, even though the final “i”CORM is less active.

This phenomenon appears to cause a misconception in many studies that relate CORM activity solely to the released CO. For example, it was suggested that the molecular basis of CORM-2 activity as procoagulant is based on CO binding to a fibrin associated heme [23], while the coagulant activity of fibrin depends on transformation of hydrophilic fibrinogen to the hydrophobic fibrin [24]. The above observations combined with those presented here, suggest that ruthenium-containing core molecules confer to CORM-2 its procoagulant properties.

It should be emphasized that the current study findings are not antagonistic to the pharmacological use of CORMs. In contrast, the outcome of the study suggests that both CO and the complementary part of CORM-2 may be physiologically active. These two parts demonstrate completely different activities: CO interacts specifically with divalent transition metals, while the core molecule acts via non-specific hydrophobic interactions. While the same CO gas is liberated from all types of CORMs and only the kinetics of its release is evaluated, the chemical nature of the complementary core molecules may by hydrophobic, amphipathic or charged and therefore, each should be studied for positive/negative side effects independently.


This study was supported by hematological research fund in memory of Kravitz family (to Mati Shaklai). The authors would like to thank Y. Goldman for valuable editorial assistance.


  1. Ryter, S.W. and Choi, A.M. (2009) Heme oxygenase- 1/carbon monoxide: From metabolism to molecular therapy. American Journal of Respiratory Cell and Molecular Biology, 41, 251-260. doi:10.1165/rcmb.2009-0170TR
  2. Gozzelino, R., Jeney, V. and Soares, M.P. (2010) Mechanisms of cell protection by heme oxygenase-1. Annual Review of Pharmacology and Toxicology, 50, 323-354. doi:10.1146/annurev.pharmtox.010909.105600
  3. Motterlini, R., Clark, J.E., Foresti, R., et al. (2002) Carbon monoxide-releasing molecules: Characterization of biochemical and vascular activities. Circulation Research, 90, 17-24. doi:10.1161/hh0202.104530
  4. Dong, D.L., Chen, C., Huang, W., et al. (2008) Tricarbonyldichlororuthenium (II) dimer (CORM2) activates non-selective cation current in human endothelial cells independently of carbon monoxide releasing. European Journal of Pharmacology, 590, 99-104. doi:10.1016/j.ejphar.2008.05.042
  5. Wilkinson, W.J. and Kemp, P.J. (2011) The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors. Purinergic Signalling, 7, 57-64. doi:10.1007/s11302-010-9213-8
  6. Nielsen, V.G., Kirklin, J.K. and George, J.F. (2009) Carbon monoxide-releasing molecule-2 increases the velocity of thrombus growth and strength in human plasma. Blood Coagulation & Fibrinolysis, 20, 377-380. doi:10.1097/MBC.0b013e32832ca3a3
  7. Deshane, J., Chen, S., Caballero, S., et al. (2007) Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism. The Journal of Experimental Medicine, 204, 605-618. doi:10.1084/jem.20061609
  8. Motterlini, R. and Otterbein, L.E. (2010) The therapeutic potential of carbon monoxide. Nature Reviews Drug Discovery, 9, 728-743. doi:10.1038/nrd3228
  9. Antonini, M.E. and Brunori, M. (1971) The derivatives of ferric hemoglobin and myoglobin. North-Holland Publishing Company, Amsterdam.
  10. Ueno, R., Shimizu, T., Kondo, K., et al. (1982) Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins. The Journal of Biological Chemistry, 257, 5584-5588.
  11. Hrkal, Z., Cabart, P. and Kalousek, I. (1992) Isolation of human haemopexin in apo-form by chromatography on S-Sepharose Fast Flow and Blue Sepharose CL-6B. Biomedical Chromatography, 6, 212-214. doi:10.1002/bmc.1130060412
  12. Tsemakhovitch, V.A., Bamm, V.V. and Shaklai, N. (2005) Vascular damage by unstable hemoglobins: The role of heme-depleted globin. Archives of Biochemistry and Biophysics, 436, 307-315. doi:10.1016/
  13. Hirota, S., Azuma, K., Fukuba, M., et al. (2005) Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere. Biochemistry, 44, 10322-10327. doi:10.1021/bi0507581
  14. Prabhu, N.P., Kumar, R. and Bhuyan, A.K. (2004) Folding barrier in horse cytochrome c: Support for a classical folding pathway. Journal of Molecular Biology, 337, 195- 208. doi: 10.1016/j.jmb.2004.01.016
  15. Bunn, H.F. and Jandl, J.H. (1968) Exchange of heme among hemoglobins and between hemoglobin and albumin. The Journal of Biological Chemistry, 243, 465-475.
  16. Liem, H.H., Spector, J.I., Conway, T.P., et al. (1975) Effect of hemoglobin and hematin on plasma clearance of hemopexin, photo-inactivated hemopexin and albumin. Proceedings of the Society for Experimental Biology, 148, 519-522.
  17. Paoli, M., Anderson, B.F., Baker, H.M., et al. (1999) Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-pro-peller domains. Nature Structural & Molecular Biology, 6, 926- 931. doi:10.1038/13294
  18. Beaven, G.H., Chen, S.H., d’Albis, A. and Gratzer, W.B. (1974) A spectroscopic study of the haemin-human-serum-albumin system. European Journal of Biochemistry, 41, 539-546. doi:10.1111/j.1432-1033.1974.tb03295.x
  19. Zunszain, P.A., Ghuman, J., Komatsu, T., et al. (2003) Crystal structural analysis of human serum albumin complexed with hemin and fatty acid. BMC Structural Biology, 3, 6. doi:10.1186/1472-6807-3-6
  20. Yang, Z., Philips, J.D., Doty, R.T., et al. (2010) Kinetics and specificity of feline leukemia virus subgroup C receptor (FLVCR) export function and its dependence on hemopexin. The Journal of Biological Chemistry, 285, 28874-28882. doi:10.1074/jbc.M110.119131
  21. Baroni, S., Mattu, M., Vannini, A., et al. (2001) Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study. European Journal of Biochemistry, 268, 6214-6220. doi:10.1046/j.0014-2956.2001.02569.x
  22. Valerie, N.G. (2009) Chemical-associated artifacts. Blood, 113, 4487.
  23. Nielsen, V.G., Cohen, J.B., Malayaman, S.N., et al. (2011) Fibrinogen is a heme-associated, carbon monoxide sensing molecule: A preliminary report. Blood Coagulation & Fibrinolysis, 22, 443-447. doi: 10.1097/MBC.0b013e328345c069
  24. van Oss, C.J. (1990) Surface properties of fibrinogen and fibrin. Journal of Protein Chemistry, 9, 487-491. doi: 10.1007/BF01024625


*This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Elena A. Sher, Sackler Faculty of Medicine, Tel-   Aviv University, Israel.

#Corresponding author.

Journal Menu >>