N. M. Senozan California State University Long Beach, 90840
Vanadium in the Living World
At the bottom of the Bay of Sevastopol in the Black Sea, in each square mile there is 400 kg of vanadium.' The metal is not locked in rocks or dispersed in silt but is in the blood and tissues of a sedentary animal called Ascidiella mpersa ( I ) . A. aspersa is a member of a group of curious animals known as tunicates. They are common to all seas but have no fresh-water or land relatives. In the phylogenetic tree they belong somewhere between vertebrates and invertebrates; a t least sometime during their life cycle they possess a primitive backbone (2). The word tunicate comes from the nonliving cellulose like substance (tunic) that envelopes these animals. Some tunicates are microscopic; some, like A. aspersa, attain a length of several inches and weigh up to 40 g. Among the three classes that make up the tunicates, vanadium occurs only in Ascidiacea (sea squirts). Most members of this class concentrate vanadium above the levels found in other marine organisms, but the accumulation reaches dramatic proportions only in the hlood cells of one family, Ascidiidae (3). It was in one of the species of this family, Phallusia mammillata, that vanadium was first discovered by German physiologist M. Henze in 1911 (41. Nearly 11% of a dark blue substance precipitated from the hlood of P. mammillata, Henze reported, was vanadium (5). Vanadocytes, the greenish vanadium carrying hlood cells of sea squirts, also contain high concentrations of sulfuric acid. The titration against an alkaline solution
has shown that when a 10-ml portion of P. mammillata mole of hydrogen ions are blood is cytolized 2.2 x produced (6). Since there are about 3.8 x loT vanadocytes/ml of mammillata blond and the volume of each vathe H+ concentration nadocyte is about 3.20 x lO-l"ml, inside a vanadocyte must be 1.83 mole/l (61. This is equivalent to a 9% solution of HzSOI. HOWmuch of this acid is in the free state is not known, but the tests on intact vanadocytes have shown the p H within the cells to be very low (6). The oxidation state of vanadium inside the vanadocytes is 3+ and undoubtedly the high acidity is essential in keeping the metal a t this low valency (7, Henze's Solution
The solution produced from the cytolysis of vanadocytes is known as Henze's solution. At p H = 2.5 Henze's solution is brownish red. When left in air it is oxidized slowly and a dark blue substance ~ r e c i ~ i t a t eIts . was in this mecipitate that Henze found 11% vanadium (51. A iater analysis of the same substance bv Webb revealed 5.6% vanadium and a still later work b; Bielig and coworkers up to 10% (6, 71. Apparently Webb's dark blue precipitate
lThroughout the teat the amounts of vanadium refer to the elemental vanadium. Although the potential for the oxidation of trivalent vanadium by oxygen increases with increasing H+ concentration, the rate of oxidation is suppressed in low pH.
Respiratory Pigments*
Piement Hemoglobin
Metal
Color
Source
Fe
Red
Mammals Birds Fish Insect Chimnomus Snail (Planorbis) Lugworm (Arenicola) Earthworm (Lumbricus) Marine worm Spimgraphis Marine warm Golfingia Octopus Lobster (Homnnls) Snail (Helix) Sea squirts
Red
Chlorocruorin
Fe
Green
Hemerytbrin
Fe
Violet
Hemocyanin
Cu
Green
Hemavanadin
V
Faint green
Number At saturation of metal number of atoms/ 0% molecules/ molecule metal atom
Location of oiement
Mol wt
Corpuscles Corpuscles Corpuscles
6.7 x l o 4 6 . 7 X 10" 6.7 X 10"
4 4 4
Plasma
3.4
lo"
2
Plasma
1.5 X 10"
96
Plasma
3 . 0 XlO"
192
Plasma
3.0
lo6
192
Plasma
2.75 X
lorb
72=
Corpuscles Plasma
1 . 0 7 X loSd 2.8 X 10"
112
Plasma
7 . 5 X 10"
20
Plasma Corpuscles
8 . 9 X 10" 2.4 X lo6(?)
X
X
Half saturation pressure (mm Hel 27 (man) 58 (chicken) 18 (salmon)
lfjd
360 24(?)
Unknown
2
*Unlessotherwise indicated the information in this table has been gathered from references (35-40). bReference (41). "Reference (42). dReference (43). Volume 51, Number8. August 1974 / 503
had some protein mixed with it which lowered the relative vanadium content (9). Most of the chemical work concerning vanadium in sea squirts has been done with Henze's solution. The results have seldom been checked and a t times have been irrepreducible even by the investigators from the same lahoratories. They have been reported mostly in biology journals and escaped the attention of chemists. The following is a short account of the findings of various workers to date. The brownish red color of Henze's solution is believed to he due to a complex of the form [V(S04)zL]3-, in which the vanadium is in its 3+ oxidation state (10). L is an organic ligand with the empirical formula ClaH1.rN3011 (11). It occupies four of the six positions around the vanadium. Electrophoresis and electrolysis experiments show that [V(S0&LI3- is in equilibrium witb S % - , LZ-, and V ( H Z O ) ~(10). ~+ A protein separates from Henze's solution when it is subjected to dialysis or ultracentrifugation or when its p H is lowered to 1 (10, 12-14). The protein is assumed to be bonded to the vanadium complex but there is no concrete evidence for this (9). The bonds-if they exist a t all-must he very weak; otherwise the metal would not separate from the protein by dialysis or ultracentrifugation. The molecular weight of the protein with vanadium associated is 24400 & 1900 (15). I t contains 11% nitrogen and 4.58% vanadium and consists of 1 part acidic, 1 part basic, and 3 parts neutral amino acids (7, 12). According to the weight analysis there must be 24 vanadium atoms per protein molecule (15). A comparison of the five known respiratory pigments with hemovanadin, a name invented for the presumed vanadium-protein compound, is given in the table. Many questions regarding Henze's solution remain unanswered. What is L? We know nothing about it except the empirical formula which has not been checked and confirmed. What is the formula and structure of the dark blue substance? In what form does vanadium exist inside the vanadocytes? I t certainly can not he [V(S04)2L]3-,for this is a brownish red species; whereas the color of a vanadocvte is light ereen. Ascidians3 are admittedlv not as important asVhu&ans; nevertheless, it is strange that our knowledge about the vanadium ~ i e m e n tbe so limited in an age when the spatial position'o? every atom in the hemoglobin molecule is known. The Function of the Vanadium
When vanadium was discovered in sea squirts it was thought to have a respiratory function. Experimental evidence did not bear this out and the idea was abandoned. The ascidian blwd did not dissolve anv more oxveen - than plain sea water; the vanadium compound did not seem to combine with oxygen reversibly (6). While the amount of oxygen absorbed-by the crab hernocyanin increases nearly four-fold when the pressure is raised from 2 to 6 mm Hg (16). nothing like it happens witb hemovanadin-so it was thought until 1968. Twenty years after his original discovery of vanadium Henze advanced a fantastic idea: The vanadium compound, being a strong reducing agent, might be involved in the synthesis of the tunic, the cellulose-like mantle, from COz (5). The idea was acknowledged to be ingenious, but was not received seriously. "Why should the ascidians need vanadium," the objection was raised, "when other tunicates can very well manufacture their mantle without it?' (6). As we shall discuss below, Henze's idea turned out to be not so far fetched; vanadium is indeed involved in the synthesis of the tunic. Bias in physical science sometimes seems to serve a useful function. Although all experiments pointed to the contrary, the belief that the vanadium must have some respiratory function persisted (1, 17). There was a compulsion to assign a hemoglobin- or hemocyanin-like role to
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504
/ Journal of Chemical Education
the vanadium compound, as is evidenced from the coinage of the word hemovanadin (12). So once again in 1968 the oxygen absorbing ability of the ascidian blood was under investigation (18). Attributing the negative results of the earlier investigators to damaged cells, Carlisle prepared a suspension of vanadocytes from P mammillata taking great care not to rupture them. And indeed, he found that the vanadocytes absorbed oxygen reversibly, becoming fully saturated a t a pressure of 5 and half saturated at 2 mm He. These values are not unusual. Manv invertebrate pigme& have a comparable or even greatkr affinity for oxygen. The flatworm Ascaris, for instance. becomes fullv saturated with oxygen at a pressure of about 0.1 mm (19). Carlisle does not mention how much gas is taken up a t saturation: so we do not know the number of moles of oxygen absorbed permole of vanadium. The members of the Ascidiidae family in which the presence of vanadium is most evident are solitary animals living often in coastal waters and rock pools where they are often exposed to the air. When the tide recedes the animal tightly closes its two syphons, the only communication it has with the outside world, and relies on the oxygen of the water entrapped in the pharyngeal cavity. On a hot summer dav alone the southern shores of Devonshire. England, the oxygen pressure inside the pharyngeal cavity is about 150 mm Ha immediately after the retreat of the tide (18). In an hour it drops to 20 mrn and after two hours to about 4 mm. Under this condition of low pressure vanadocytes can efficiently transport oxygen to the tissues. Yet it is difficult to believe that the vanadocytes evolved only to serve the ascidians living on the edge of tide waters on hot summer days. I t would have been far easier for these animals to move where they could bathe themselves continuously and get enough oxygen from the sea water, rather than to develop a complex chemical. More likely nature picked vanadium for another purpose and those ascidians livine in littoral zones haonened to find a secondary use for i< This thesis is supportld by the work of Endean who discovered that the svnthesis of the fibers from which the tunic is built starts within the vanadocytes (20, 21). How these fibers are synthesized and what role vanadium plays are unknown; perhaps it indeed is the carbon dioxide that is reduced by hemovanadin to the cellulose-like molecules of the tunic, as Henze had proposed many years before.
~g
The Source of the Vanadium
An adult P. mammillata contains about 8 mg of vanadium, 85% of which is in vanadocytes (6). The source of this vanadium has long been a mystery. It has been reported, in chronological order, that the sea water has 0.3-0.6, 0.1, 3-4, 2, and 0.02 mg of vanadium/m3 (22-26). Then to accumulate 8 mg of metal the sea squirt must process somewhere between 2-400 X 103 1 of water. It takes 5 yr for the mammillata to reach maturity; so every minute 1-200 ml of water must be filtered with 100% efficiency. In Ciona intestinalis, a common ascidian that fouls the bottoms of barges and docks.4 the efficiency of vanadium uptake is 2.5% (23). If the same efficiency is assumed for P. mammillata we come to the conclusion that the animal must process 40-8000 ml of water every minute. Even the lower figure is suprising for a fist-size organism. Vinogradov suggests that ocean silt which contains 10-2-10-3% vanadium might be the source of the metal (27). To support his contention he points out that the vanadium accumulation occurs only in sedentary ascidians. This is a plausible idea, though i t is difficult to understand how a sea squirt can digest ocean silt and extract "scidian (sea squirt) is the adjective for the species in the class afAseidiacea. C. intestinalis has been suggested as a possible commercial source of special grade vanadium (31).
the vanadium from it. The boiling of mud from a region where P. mammillata was abundant, with 5% hydrochloric acid for 2 hr, did not release a detectable amount of the metal (6). As a third possible source Bertrand mentions a vanadium rich plankton (3). hut no such creature has yet been found. Certain ascidians outside the Ascidiidae family do not have vanadocytes, yet accumulate substantial amounts of vanadium. C. intestinalis, that common infester of piers and barges, is an example of such an ascidian. Elsewhere in the animal world vanadium does not occur except in traces.5 In the plant kingdom one species collects the metal in striking amounts: Amanita muscaria, a brightly colored, deadly mushroom (28, 29). Vanadium is 0.02% of its dry weight. Neither in ascidians that do not have vanadocytes nor in A. muscarin is anything known about the function or the chemical nature of the vanadium. One final note: Whatever the source of their vanadium, different ascidians seem to have a different preference for the isotopes of this metal. The 5oV/51V ratio changes by as much as 4% from one animal to another 130). This is in sharp contrast with the composition of inorganic vanadium sources, where even the highest resolution mass spectrometers have not been able to detect a variation in the isotopic constitution (30). Literature Cited 11) Vinogradw, A. P.. "The Elementary Chemical Compaeicion of Marine Organisms," Seam Foundation lor Marino Research. Yale Univenily. New Haven. I9D.p427. 121 Barnes. R. D., "Invertebrate Zoology," 2nd Ed.. W. B. Ssunders Company. Philadelphia. 196%p 681. (3) Bertrand. D.. Hull. Amer M u . Nat. Hi&. 94,407119501. (4) Henre. M.,Hoppa-S~yler'sZ.Physioi. Chem.. 72,494 (19111. (5) Henze.M.. Happe-Seylw'8Z. Physioi. Chem.. 213. 125(1932l. 16) Webb. D.A.. J E z p . Biol.. 16,499(19391. (7) Biolig, H. .I.. Bayer, E., Califsno. L.. and Wirth. L., Pubbl. Sfoz. Zoal. Nc%poli.25,
*"\.""-,. +e,,oc",
18) Relaova, L.T.. Zh. Obrhrh. B i d 25.3471(19MI, cf., C.A. 62.3129h 119651. (91 Webb,O.A..Pubbl.Slal. Zoo1 Napoli, 2R,n311956l. (lo1 Bielie. H. J.. Doll. H. 0 . Mallinter. H., and Rudige, W.. Jvstua Licbigr Ann. ~ L r n . 662. . 206(19631. (111 Mollin~er,H.. PhD. Thesis, Univ. ofFreiburp. Germany. 1960. (12) Califsno. L.. andcaselli. P.,Pubbl. Sfoz.Zoa1. Nopoii. 21.261 (19491.
(14, B ~ I G ~H.,~a nC d ~ ~e &~i a .i.. ~ f~i b h, i . Stol. ZOOI ~ o p o i i30, . ix (19581. (16) Biolig, H. J.,andBsyer, E.. Ezparirniio, 10.3W (19541. (16) Rodmond. J. R.. in "Physiology and Biochemistry of Hemowanin." (Ediloi: Ghir~ etti,F.),AcadrmicPxss, Inc.. London. 1968,pll. I171 Vallee. B. L.. and Wackor. W. E. C.. in "The Pmteins." (Editor: Neurath, H.). . ~ o i ~ o r1970. k , p39. ~ m d ~ mPi c~ Whe.. (L8) Cadisle, D.B..Proc. Roy Soe., S e r 8. 171,31 (19681. (19) Pmsser, C. L.. end Brouln. F. A., .Jr. "Camparative Animal Physiology," W. B. Saunden Company, Philsdclphia. 1181. p 211. (201 Endean. R., Quart. J. Microse. Sci., 101.177 119601. (21) Endean,R., Quofi. J Microsc S r i , 102,107119611. (22) Ernst. T.. and Hormsnn. H.. Norhr. G.8. Wiss.. Gnfingm. Fachpuplle IV. 1.205
127) Ref. (1). pp427-8. (281 Beinert. H.,andPalmer, G.,Aduan. Eniymol.. 27.105(1965). (291 Wetkinson, J . H., Nature. 202.1239 l19Ml. 3. 571 (30) Fleaeh. G. 0.. Capellen. J.. and SXC, H. J., Admn. Mas*. SPOCLID~..
,,-, ,.""",.
I311 Elroi. D.. and Komamvsky, B., Den. F k k r i a Council Maditwmnean, Pmc. Tech. PnnnraNo ..-. ~. L ~
261 (196li ~~
(321 Phillips, A. H..Arnsr.J. Sci.. 46.173 (19181. 1331 cierwzko. L. S.. Cicresrko. ti. M.. Hanis. E. R.. and Lane. C. A.. cam^ Bio-
...,"...., .
,.""-,.
I341 Webb. D.A.. Sci. Pmc. R. DubljnSoc.. 2l,505(19371. I351 donor, .I. D.. "Comparative Phy~iologyof Rerpirafion." Edward Amdd Lld.. London, 1912. (361 Manuell. C.. in ''Oxygen in the Animal Organism," (Edilon: Dickens. F.. and Neil. E1,TheMacMiilanCompany. New York. 1961. (371 PC-r. C. L.. and Bmwn, F. A,. Jr.. "Comparative Animal Physi~log~." W. B. '"Y. maaoe,pn,a. Ira,. I361 Baldrin. E.. "An Intmduction to Comparative Biorhemistry:'Cambridge Univevaity Press. Cambridge. 1939. (391 Manwell, C.,Ann. Re". Physiol.. 22.191 119601. (40) Wyman, J.. Jr.. Ad". Protein Chem.. 4.407 l1848I. (411 ~ & n i n i . ti., Rmsi~Fanolli. A . Capurn. A,. Arch. Biochem. Bcaphys. 97. 343
,.""&,. ,,-v>
1621 Guerrilore, D.. etsl.. J. Ma1 Biol., 13,234 (19651. (43) Klotz. I.M.. endKeresztesNagy. S..Biochrrnisl~. 2.445.923 (1963).
Substantial amounts of vanadium have been reported inStichopus mobii, a sea cucumber, by Phillips (3% but it bas not been possible to reproduce his results (33). There also exists en unconfinned report of vanadium in the mollusk Pleurobranehus plumuIn (34).
Volume 51, Number 8. August 1974 / 505
