N. M. Senozan California State University Long Beach. 90840
Hernocyanin: The Copper Blood
In the cool waters of the Humboldt Current off the coast of Peru lives a large and dangerous squid known as Dosidicus gigas. Its cigar-shaped body with tentacles included can reach to a length of 12 ft and weigh as much as 350 lhs. The powerful muscles beneath its mantle can squirt water with the force of a "fire hose" and propel the animal a t speeds exceeding 20 miihr (1-2). Dosidicus is a ferociouspredator. Its beak is sharp enough to cut through a steel cable and i t has been seen to shred large fish weighing over 50 lbs intopieces. "A man who fell over board in waters where these animals abound," it has been said, "would not last half a minute" (3). T o maintain such vigorous activity and savage existence, the squid of the Humholdt Current requires some 50 1 of oxygen1 an hour which is carried to its tissues by a copper containing protein molecule called hemocyanin. The name is misleading. Hemocyanin, unlike hemoglobin, has no heme group; the copper is bound directly to the protein. The hinding is strong; the dissociation constant for the re( 4 ) .The moval of the first copper atom is in the order of site of the hinding is not known, although on circumstantial evidence the sulfhydryl groups of the amino acid cysteine and the imidazole rings of histidine have been suggested as possible locations for the metal (5.6). In oxygen free hemocyanin the copper is in the 1+oxidation state. There are several pieces of evidence for this. Chelating agents specific for Cu+ can remove the metal from the protein, and physiologically active hemocyanin molecules can he reconstituted when the protein from which the copper has been removed is treated with cuprous ions (7, 8). In the uv and visible spectrum there are no characteristic cupric hands and, finally, no esr signal has ever been detected in the deoxygenated pigment (indicative of d l o configuration) (9). In oxyhemocyanin, however, the situation is very confusing. Biquinoline, a reagent that gives a distinctly pink complex with cuprous ions, reacts with one-half of the copper in hemocyanin suggesting that half the metal in the pigment is in the 1+and the remainder in the 2+ state (5). In the uv and visihle spectrum there are characteristic cupric bands which again suggest that at least some of the copper is in the 2+ state (10). The difficulty arises in interpreting the esr spectrum. Cupric ions having an odd number of electrons should show a strong esr absorption. Yet freshly prepared oxyhemocyanin shows no such absorption (11). Despite the absence of an esr signal Professor H. B. Gray of the California Institute of Technology believes that copper in oxyhemocyanin is in the cupric state (12). He attributes the lack of a n esr signal to an antiferromagnetic coupling between two copper atoms. It is also conceivable that the metal is part of a covalent frame in which its behavior just happens to resemble cuprous ions in some instances, cupric in others. The Occurrence ol Hemocyanin
Hemocyanin occurs in only two phyla of animals, mollusks and arthropods, as shown in Figure 1. Among arthropods it is found in all decapod crustaceans (lobsters, crabs, shrimps) and among mollusks in all cephalopods (octopus, squid, cuttlefish) (9,13-14). Elsewhere in these two phyla, however, the distribution of hemocyanin is erratic. The edible snail "escargot" contains large quantities, hut its fresh-water relative Planorbis has none. While clams, oysters, scallops, and other 684 / Journal of Chemical Education
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Volume 53, Number 11. November 1976 / 687
state of aggregation also depends on the extent of oxygenation (37). Both fully oxygenated and fully deoxygenated squid hemocyanin, they observed, exist as 59s aggregates, hut around 80%oxygenation the pigment breaks entirely into 20 and l l S units. The behavior of the gastropod hemocyanins is somewhat different in this respect.instead of breaking into smaller units first, then associating back, the aggegation state of these molecules tends to change in only one direction. As the oxygenation progresses they either dissociate into smaller units ;;aggregate into larger blocks (48,49). The fact that cooperativity and aggregation are often brought about simultaneously by the presence of divalent ions implies that there might he a connection between the two nhenomena. The effect of oxveenation on the aemeeation ... .. *statealso points at such a connection. Two Dutch biochemists, van ihiel and van Brueeen.. have in fact calculated a simoidal dissociation curve for the H. pomatia hemocyanin using the exoerimentallv determined concentrations of 605 and 205 unitsat variousdegrees ofoxygenation and assuming that the 205 unit has a half saturation oressure of 4 mm He. and the 6US unit 40 mm Hg (48). It now appears, however, that the explanation of cooperativity on the basis of association or dissociation alone is inadequate. Erza Daniel and his coworkers from Tel-Aviv University note that the ratio of 205 to 100s units remains unchanged as the hemocyanin of snail Leuantina hierosolima is oxygenated (50). Furthermore, sigmoidal dissociation curves are obtained even when all the hemocyanin in the solution is in 20 or 100s form. These observations clearly rule (nit the possibility that the aggregation or dissociation are the only mechanisms for coopera&&y. A hemocyanin molecule, it is now believed, can exist in two conformationally distinct forms (35,36,39,51). These forms have different affinities for oxygen. Deoxyhemocyanin molecules are all in the low affmitv conformation. At intermediate saturations both forms are present and in fully aerated solutions the high affinitv form ~redominates.The coo~erativitv according Gthis simGle and kndouhtedly approxim& pi&& is attributed to the formation of . oroeressivelv . lareer amounts ofhigh affinity rnulec.ules as the oxygenation proceeds. Since sigmoidd curves arc t,hsewed only in the presence of Ca2' and Mg", we must conclude that divalent ions are essential for theexistence of twodistinct cmformations. Just how adivalent ion makes it possible for a hemocyanin molecule to acmire two distinct conformations with widelv varvina oxveen affinities is one more of the mysteries surrounding'thg ancient pigment.
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Literature Cited (11 Clarke, M. R..Adu.Mor. Bioi., 4.93 (1966). 121 Lane, W. F., "Kinpdom of the Octapus;' Pyramid Publications, 1nc.. New Yurk.
688 / Journal of Chemical Education
(8) Lontie, R., Blaton. V., Albcri. M., and Peetam. B.. Arch. Inf. Physiol. Biochim.. 73.
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(91 Van Holde. K. E., andvan Bruggen, E. F. &.in "Subunitn in BiolagicelSystams,Psrf A," IEditora: Timaahdf, S. N.. and Fasmsn, G.D.1, M a r 4 Dekker. Inr., New York,
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(10) Van Holde, K. E.. RiackemMry, 6.93 11967). i l l ) Naksmura.T..andMason, H.S.,Biocham.Riophya. Re$. Commun.. 3.297 (19601. 112) Gray, H 8..in "~ioinorgsni~Chemirtry."(Editoc G d d . R.F.l.Am~rieanChemica1 Society. Washington, 1971. (131 Ghirefti, F., in "Oxygenases," (Editor Hayaishi. O.),AcsdemicPras~.Ine., New Yurk. '&C" .""a.
114) Vinogradou. A. P., '"The Elementary Chemical Composition of Marine Organisms." Sears Foundation for Marine Reearch, Yak Univenify, New Haven. 1953, p. 149. (151 Pi1son.M. E.Q..Bioi. Bull., 128.459i1965). 116) Manwell, C..Ann. Rw~Physioi.,22,191 (19601. (17) Redfield. A. C.. Biol Rev., 9.175 119341. 118) Ellerton. H. D.. Carpente~D. E.. and van Hulde. K. E., Biochemistry, 9. 2226
