**7. Conclusions**

Although carbonic anhydrase represents one of the most investigated metalloenzyme in nature, its interaction with heavy metals has been only partially elucidated to date and some issues still remains to be explored. An intriguing aspect that needs more investigation is the *in vivo* effect of heavy metals on CA expression. From the few studies available in literature some metals appear to be important modulator of the expression of this protein. The understanding of the underlying mechanisms could open new perspective in the comprehension of the functioning and regulation of this enzyme. Another intriguing aspect of the biochemistry of CA is the inhibition by heavy metals. It has been documented for some species and some metals, but the mechanisms behind the inhibition, its metal specificity and isoform specificity remains still unknown. These aspects merits in depth examination and open new perspective for drug design and biomarkers development.

#### **8. References**

Adams, S.M.; Crumby, W.D.; Greeley, M.S.; Ryon, M.G. & Schilling, E.M. (1990). Relationship between Physiological and Fish Population Responses in a Contaminated Stream. Environmental Toxicology and Chemistry, Vol. 11, Issue 11, (November 1992), pp. 1549-1557, ISSN: 0730-7268

adjunct model to whole-animal *in vivo* exposure and to ecotoxicological evaluation of the potential risk of trace pollutants in aquatic environments. They are rapid, low cost and simple tools to be utilized in combination with chemical analysis, for the pre-screening of the environmental samples that should be analyzed. Lionetto et al (2005; 2006) explored the possible application of heavy metal CA inhibition for the development of an *in vitro* bioassay applicable to the determination of the toxicity of environmental aqueous samples. They developed rapid and sensitive chemical hazard detection system for standardizing

In the last years the affinity of carbonic anhydrase for metal ions has been applied for the development of fluorescence based biosensors for determination of free metal ions in solution using variants of human carbonic anhydrase (apoCA). In particular, Cu2+, Co2+, Zn2+, Cd2+, and Ni2+ have been determined at concentration down the picomolar range (Fierke and Thompson, 2001; Thompson and Jones, 1993; Mey et al., 2011) by changes in fluorescence emission (Thompson et al., 2000) and excitation wavelength ratios (Thompson et al., 2002), lifetimes (Thompson and Patchan, 1995), and anisotropy (polarization) (Elbaum et al., 1996; Thompson et al., 2000). The sensitivity, selectivity, analyte binding, kinetics and stability of the biosensors have been improved by subtle modification of the protein structure by directed mutagenesis (Kiefer et al., 1995; Hunt et al., 1999; DiTusa et al., 2001; McCall et al., 2004; Burton et al, 2000). These studies have hallowed the development of highly selective and sensitive fluorescence-based biosensors for Zn2+ e Cu2+, which have been shown to be viable approach in some important applications. In fact, the CA-based Cu2+ biosensor has been used to obtain real-time measurement of free Cu(II) at picomolar concentrations in seawater (Zeng et al., 2003), while the CA-base Zn2+ biosensor has been used for measurement of free Zn ion at

Although carbonic anhydrase represents one of the most investigated metalloenzyme in nature, its interaction with heavy metals has been only partially elucidated to date and some issues still remains to be explored. An intriguing aspect that needs more investigation is the *in vivo* effect of heavy metals on CA expression. From the few studies available in literature some metals appear to be important modulator of the expression of this protein. The understanding of the underlying mechanisms could open new perspective in the comprehension of the functioning and regulation of this enzyme. Another intriguing aspect of the biochemistry of CA is the inhibition by heavy metals. It has been documented for some species and some metals, but the mechanisms behind the inhibition, its metal specificity and isoform specificity remains still unknown. These aspects merits in depth examination and open new perspective for drug design and biomarkers development.

Adams, S.M.; Crumby, W.D.; Greeley, M.S.; Ryon, M.G. & Schilling, E.M. (1990).

(November 1992), pp. 1549-1557, ISSN: 0730-7268

Relationship between Physiological and Fish Population Responses in a Contaminated Stream. Environmental Toxicology and Chemistry, Vol. 11, Issue 11,

rapid, sensitive, and low cost CA based *in vitro* bioassay (Schettino et al., 2008).

**6. Carbonic anhydrase-based biosensing of metal ions** 

picomolar levels in cultured cells (Bozym et al, 2004).

**7. Conclusions** 

**8. References** 


Cox, J.D.; Hunt, J.A.; Compher, K.M.; Fierke, C.A. & Christianson, D.W. (2000). Structural

De Mora, S.; Fowler, S.W.; Wyse, E. & Azemard, S. (2004). Distribution of heavy metals in

DiTusa, C.A.; McCall, K.A.; Chritensen, T.; Mahapatro, M.; Fierke, C.A. & Toone, E.J. (2001).

Elbaum, D.; Nair, S.K.; Patchan, M.W.; Thompson, R.B. & Christianson, D.W. (1996).

Esbaugh, A.J. & Tufts, B.L. (2006). The structure and function of carbonic anhydrase

Ferry, J.F. (2010). The gamma class of carbonic anhydrases*. Biochimica et Biophysica Acta* 

Fierke, C.A. & Thompson, R.B. (2001). Fluorescence-based biosensing of zinc using carbonic

Gilbert, A.L. & Guzman, H.M. (2001). Bioindication potential of carbonic anhydrase activity

Goto, T.; Shirakawa, H.; Furukawa Y. & Komai, M. (2008). Decreased expression of carbonic

Grimes, A.; Paynter, J.; Walker, I.D.; Bhave, M. & Mercer, J.F.B. (1997). Decreased carbonic

Heisler, N. (1984). Acid–base regulation in fishes. In: *Hoar*, W.S., Randall, D.J. (Eds.), Fish

Physiology, vol. 10A. Academic Press, New York, pp. 315–401.

295, Lewis Publisher, ISBN 978-0873716482, Boca Raton, USA

*Medicinal Chemistry*, Vol.22, Issue 6, pp.745–750, ISSN 1475-6366

Vol.239, Issue 1, (July 1986), pp. 17–23, ISSN 0022-104X

13694, ISSN 0006-2960

1570-9639

ISSN 0264-6021

pp. 742–744, ISSN 0025-326X

2008), pp. 248-53, ISSN 0007-1145

0844

Influence of Hydrophobic Core Residues on Metal Binding and Specificity in Carbonic Anhydrase II. *Biochemistry*, Vol.39, No.45, (November 2000), pp.13687–

marine bivalves, fish and coastal sediments in the Gulf and Gulf of Oman, *Marine Pollution Bulletin,* Vol. 49, Issue 5-6, (September 2004), pp.410-424, ISSN 0025-326X Depledge, M.H. (1994). The rational basis for the use of biomarkers as ecotoxicological tools.

In: *Nondestructive Biomarkers in Vertebrates*, M.C., Fossi; C., Leonzio (Eds.), pp. 271–

Thermodynamics of metal ion binding. 2. Metal ion binding by carbonic anhydrase variants. *Biochemistry*, Vol.40, Issue 18, (May 2001), pp.5345-5351, ISSN 0006-2960 Ekinci, D.; Beydemir, Ş. & Küfrevioğlu Ö.İ. (2007). In vitro inhibitory effects of some heavy

metals on human erytrocyte carbonic anhydrases. *Journal of Enzyme Inhibition and* 

Structure-based design of a sulfonamide probe for fluorescence anisotropy detection of zinc with a carbonic anhydrase-based biosensor. *Journal of the American Chemical Society*, Vol. 118, Issue 35, (September 1996), pp. 8381-8387, ISSN 0002-7863

isozymes in the respiratory system of vertebrates. *Respiratory Physiology & Neurobiology*, Vol.154, Issue 1-2, (November 2006), pp. 185–198, ISSN 1569-9048 Evans, D.H. & Cameron, J.N. (1986). Gill ammonia transport. *Journal of Experimental Zoology*,

*(BBA) - Proteins & Proteomics*, Vol.1804, Issue 2, (February 2010), pp. 374-38, ISSN

anhydrase. *Biometals*, Vol. 14, Issue 3-4, (September 2001), pp. 205-222, ISSN 0966-

in anemones and corals. *Marine Pollution Bulletin*, Vol. 42, Issue 9, (September 2001),

anhydrase isozyme II, rather than of isozyme VI, in submandibular glands in longterm zinc-deficient rats. British Journal of Nutrition, Vol.99, Issue 2, (February

anhydrase III levels in the liver of the mouse mutant "toxic milk" (tx) due to copper accumulation, *Biochemical Journal*, Vol. 321, Part. 2, (January 1997), pp. 341–346,


Kuhara, M.; Wang, J.; Flores, M.J.; Qiao, Z.; Koizumi, Y.; Koyota, S.; Taniguchi, M. &

Lane, T.W. & Morel, F.M.M. (2000). A biological function for cadmium in marine diatoms.

Lane, T.W.; Saito, M.A.; George, G.N.; Pickering, I.J.; Prince, R.C. & Morel, F.M.M. (2005). A

Lee, J.G.; Roberts, S.B. & Morel, F.M.M. (1995). Cadmium a nutrient for the marine diatom.

Liang, J.Y. & Lipscomb, W. N (1988). Hydration of CO2 By Carbonic-Anhydrase –

Lionetto, M.G.; Maffia, M.; Cappello, M.S.; Giordano, M.E.; Storelli, C. & Schettino, T. (1998).

Lionetto, M.G.; Caricato, R.; Giordano, M.E.; Pascariello, M.F.; Marinosci, L. & Schettino, T.

Lionetto, M.G.; Caricato, R.; Erroi, E.; Giordano, M.E. & Schettino, T. (2005). Carbonic

Lionetto, M.G.; Caricato, R.; Erroi, E.; Giordano, M.E. & Schettino, T. (2006). Potential

*Chemistry and Ecology*, Vol. 22, Supplement 1, pp. 119-125, ISSN: 0275-7540 Lukaski, H.C. (2005). Low dietary zinc decreases erythrocyte carbonic anhydrase activities

*Clinical Nutrition*, Vol 81, Issue 5, (May 2005), pp.1045–1051, ISSN 0002-9165 MacAuley, S.R.; Zimmerman, S.A.; Apolinario, E.E.; Evilia, C.; Hou, Y.; Ferry, J.G. & Sowers,

Maffia, M.; Trischitta, F.; Lionetto, M.G.; Storelli, C. & Schettino, T. (1996). Bicarbonate

Research, Vol. 32 (2), (April 2011), pp. 111-117, ISSN 0388-6107

Issue 9, (April 2000), pp. 4627–4631, ISSN 0027-8424

pp.42–42, - ISSN 0028-0836

pp. 8676-8682, ISSN 0006-2960

0024-3590

ISSN: 0166-445X

330, ISSN 0025-326X

ISSN: 0306-7319

ISSN: 0006-2960

Sugiyama, T. (2011). Sexual dimorphism in LEC rat liver: suppression of carbonic anhydrase III, by copper accumulation during hepatocarcinogenesis. Biomedical

*Proceedings of the National Academy of Sciences of the United States of America*, Vol.97,

cadmium enzyme from a marine diatom. *Nature,* Vol. 435, Issue 7038, (May 2005),

*Limnology and Oceanography*, Vol. 40, Issue 6, (September 1995), pp. 1056-1063, ISSN

Intramolecular Proton Transfer between Zn2+-Bound H2O and Histidine-64 in Human Carbonic Anhydrase-II. *Biochemistry*, Vol. 27, Issue 23, (November 1988),

Effect of cadmium on carbonic anhydrase and Na+-K+-ATPase in eel, Anguilla anguilla, intestine and gills. *Comparative Biochemistry and Physiology A- Molecular and Integrative Physiology*, Vol.120, Issue 1, (May 1998), pp.89-91, ISSN 1095-6433 Lionetto, M.G.; Giordano, M.E.; Vilella, S. & Schettino, T. (2000). Inhibition of eel enzymatic

activities by cadmium. *Aquatic Toxicology*, Vol. 48,Issue 4, (April 2000), pp. 561-571,

(2003). Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymatic activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area. *Marine Pollution Bulletin*, Vol. 46, Issue 3, (March 2003), pp. 324-

anhydrase based environmental bioassay. *International Journal of Environmental Analytical Chemistry*, Vol.85, Issue 12-13, (October-November 2005), pp. 895-903,

application of carbonic anhydrase activity in bioassay and biomarker studies.

and impairs cardiorespiratory function in men during exercise. *American Journal of* 

K.R. (2009). The archetype γ-class carbonic anhydrase (Cam) contains iron when synthesized in vivo, *Biochemistry*, Vol. 48, Issue 5, (February 2009) pp 817–819,

absorption in eel intestine: Evidence for the presence of membrane-bound carbonic

anhydrase on the brush border membranes of the enterocyte. *Journal of Experimental Zoology*, Vol: 275, Issue 5, (August 1996), pp. 365-373, ISSN 0022-104X


Randall, D.J. & Daxbaeck, C. (1984). Oxygen and carbon dioxide transfer across fish gills. In:

Roberts, S.B.; Lane, T.W. & Morel, F.M.M. (1997). Carbonic anhydrase in the marine diatom

Rowlett, R.S. (2010). Structure and catalytic mechanism of the β-carbonic anhydrases.

Schettino, T.; Trischitta, F; Denaro, M.G.; Faggio, C. & Fucile, I. (1992). Requirement of

Schettino, T.; Lionetto, M.G. & Erroi, E. (2008). Enzymatic method for the dectetion of the

Skaggs, H.S. & Henry, R.P. (2002). Inhibition of carbonic anhydrase in the gills of two

Soto, M.; Ireland, M.P. & Marigómez, I. (2000). Changes in mussel biometry on exposure to

Soyut, H.; Beydemir, Ş. & Hisar, O. (2008). Effects of Some Metals on Carbonic Anhydrase

Supuran, C.T.; Scozzafava, A. & Casini, A. (2003). Carbonic anhydrase inhibitors. *Medicinal Research Reviews*, Vol.23, Issue 2, (March 2003), pp. 146– 189, ISSN: 0198-6325 Supuran, C.T. & Scozzafava, A. (2007). Carbonic anhydrases as targets for medicinal

Supuran C.T., (2008). Carbonic anhydrases: novel therapeutic applications for inhibitors and

Supuran, C.T. (2010). Carbonic anhydrase inhibitor. *Bioorganic & Medicinal Chemistry Letters*,

Tashian, R.E.; Hewett-Emmett, D.; Carter, N.D. & Bergenhem, N.C.H. (2000). Carbonic

Thompson, R.B. & Jones, E.R. (1993). Enzyme-based fiber optic zinc biosensor. Analitical Chemistry, Vol.65, Issue 6, March 1993), pp. 730-734, ISSN 0003-2700

Vol. 20, Issue 12, (June), pp. 3467-3474, ISSN: 0960-894X

York, pp. 263–314, ISBN 0-12-350430-9

HCO3

0031-6768

PCT/EP2008/064703

pp. 175-187, ISSN 0048-9697

4350, ISSN: 0968-0896

ISSN 1474-1776

(June 2008), pp.179–190, ISSN 0163-4984

Basel, pp. 105–120, ISBN 3-7643-5670-7

(October 1997), pp. 845–850, ISSN 0022-3646

(February 2010), pp. 362-373, ISSN: 1570-9639

4, (December 2002), pp. 605-612, ISSN 1532-0456

Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. 10A. Academic Press, New

Thalassiosira weissflogii (Bacillariophyceae). Journal of Phycology, Vol. 33, Issue 5,

Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, Vol.1804, Issue 2,

tozicity of aqueous environmental matrices. Patent n. MI2008A008813,

euryhaline crabs, Callinectes sapidus and Carcinus maenas, by heavy metals. *Comparative Biochemistry and Physiology C-Toxicology & Pharmacology*, Vol. 133, Issue

metals: implications in estimation of metal bioavailability in "Mussel-Watch" programmes. *The Sciences of the Total Environment*, Vol. 247, Issue 2-3, (March 2000),

from Brains of Rainbow Trout. *Biological Trace Element Research*, Vol.123, Issue 1-3,

chemistry. *Bioorganic & Medicinal Chemistry*, Vol. 15, Issue 13, (July 2007), pp. 4336–

activators. Nature Reviews Drug Discovery, Vol.7, Issue 2, (February), pp.168–181,

anhydrase (CA)-related proteins (CA-RPs) and transmembrane proteins with CA or CA-RP domains. In: Chegwidden, W.R.; Carter, N.D. & Edwards, Y.H. (Eds.), Carbonic anhydrase (CA)-related proteins (CA-RPs) and transmembrane proteins with CA or CA-RP domains. *The Carbonic Anhydases: New Horizons*. Birkhauser,


