ABSTRACT
It has been known since 1911 (Henze) that the blood of Phallusia mamillata Cuv. contains vanadium, which is found in special cells, called vanadocytes by Webb (1939). Webb estimated that the vanadocytes constitute about 60% of the blood cells and the blood cell volume amounts to between 1 and 2% of the total blood volume. It was found by Henze that the inner medium of the vanadocytes is very acid, due to free sulphuric acid which is present in a concentration of about 3%. According to Webb, the vanadium content of the animal amounts to about 0-15% of the dry weight of the body, and three-quarters of the total amount of vanadium is present in the blood, 15 % in the gut and associated viscera and 10% in the mantle and branchial sac.
The pigment present in the vanadocytes is referred to as native haemovanadin, and the reddish brown pigment obtained following their haemolysis with distilled water as red haemovanadin following the terminology of Califano & Caselli (1947). On addition of alkali to a solution of red haemovanadin the pigment precipitates as blue haemovanadin.
The function of this vanadium compound is not known. Winterstein (1909) excluded any oxygen-carrying power (a fact which was confirmed during the present investigations). Henze (1932) found that red haemovanadin reduces methylene blue and indigo carmine, and according to Webb it reduces inorganic ferric to ferrous iron. From the reducing power Henze deduced that the vanadium should be present in the form of vanadium dioxide V2O2 (in which vanadium is bivalent), incorporated into a non-dialysable molecule, which he believed at first to be a protein though he later cast doubt on this opinion. Webb, on the contrary, found that the pigment is dialysable, and did not consider it to be a protein. Califano & Caselli (1947) found that the solution obtained following haemolysis contains a protein, from which the vanadium could be split by simple means, like ultra-filtration or dialysis. Califano & Caselli (1950) also determined the ultra-violet absorption curve of the haemolysate and of its dialysate.
In this paper particular attention has been paid to those properties which may be related to its physiological function. In addition we have demonstrated the presence of cytochrome c in sperm and in ripe eggs, and have shown that the respiration of tissues of the adult animal is cyanide-stable.
* A preliminary report of this work was presented at the First International Congress of Biochemistry, Cambridge, August 1949.
Techniques
Blood was taken from the large branchial vessel of five to ten freshly captured animals. The blood was centrifuged, the cells separated and washed either with filtered sea water or with a solution containing NaCl, KC1, KH2PO4 and MgSO4 in the same relative proportions as for Krebs-Ringer solution (see Cohen, 1945) but corrected to a molarity of 0·6. The blood cells were then haemolysed with distilled water acidified to pH 2·4 with sulphuric acid (glass electrode). The stromata of the cells were removed by centrifugation and filtration through a glass filter, and the clear solution was then dialysed overnight at 4 ° C. in a cellophane sac against distilled water acidified with H2SO4 to pH 2·4. Dialysis removed the non-colloidal components of the haemolysate, leaving a clear solution which could be stored at 4 ° C. for several days without deterioration. In fact, even after a week’s storage the reducing power of the solution usually decreased by only a few per cent.
The bivalent vanadium content of the solution of haemovanadin was determined, as described by Henze (1932), by methylene blue titration, using a millimolar solution brought to pH 2·4 with sulphuric acid.
Routine experiments for the detection of the spectrum of cytochrome c were made with a Zeiss hand spectroscope. For more accurate determinations a Beckman photoelectric quartz spectrophotometer was used.
Cytochrome c solutions were prepared from bovine heart by the method of Keilin & Hartree (1937), and their concentrations determined spectrophoto-metrically.
Catalase solutions were prepared from beef liver according to the method of Sumner & Dounce (1939).
V2O2 (lavender blue) was produced by reduction of ammonium metavanadate by zinc dust in the presence of sulphuric acid.
Results
First of all it was found that, in order to keep the vanadium compound in the state of native haemovanadin, it is essential to maintain the pH of the medium at about pH 2·4. Therefore, haemolysis was carried out with distilled water made acid with sulphuric acid, and dialysis was also conducted against acidified water at pH 2·4. At higher pH the vanadium compound inside the cellophane sac gradually precipitates as a blue pigment. At pH 2·4 the compound does not change its colour, and the vanadium which appears in the outer solution can no longer reduce methylene blue. Thus we believe that this vanadium is no longer in the bivalent form.
The reducing power of native haemovanadin towards methylene blue seemed to offer the most promising approach for a study of its function. Native haemovanadin at pH 2·4 is only extremely slowly autoxidizable ; for example, it can be stored for more than a week in the presence of air at 4 °C. At higher pH the autoxidation becomes very rapid.
The addition of ferric cytochrome c (from beef-heart), both under aerobic and anaerobic conditions (in Thunberg tubes), at the same pH as the haemovanadin solution (usually pH 2·4, but also up to pH 4·0) brings about a rapid oxidation of the haemovanadin together with a reduction of cytochrome c. The reduction of the cytochrome was followed with the spectroscope, while the oxidation of the haemovanadin was determined by titration with methylene blue. We could not follow the kinetics of the reaction spectrophotometrically, as determinations of the extinction coefficients were vitiated by a slight turbidity. Nevertheless, even by visual control it was easy to see that the reaction was rapid (it appeared complete within 2 min.) and that it was speeded up on raising the pH. For instance, the addition of a cytochrome solution of pH 3.0 to a haemovanadin solution at pH 2·4 gave quicker reduction of the cytochrome than the addition of the same amount of cytochrome which had been adjusted to pH 2·4. We assume that the haemovanadin oxidation by ferric cytochrome c is speeded up by hydroxyl ions.
Mention should be made of a phenomenon that occurred during our experiments. With some of our pigment preparations, both under aerobic and anaerobic conditions, the cytochrome c which had been reduced by haemovanadin slowly reverted to the oxidized ferric form. Under anaerobic conditions, this fact could not be attributed to autoxidation of cytochrome in the acid solution by molecular oxygen. By the addition of traces of catalase this oxidation was suppressed. We are unable to explain the phenomenon and the eventual presence of hydrogen peroxide in the reaction.
The stoichiometric ratio of the reaction was determined as follows: a standard solution of cytochrome c containing 0·0177 mmol, of ferric cytochrome c per litre was titrated with a haemovanadin solution containing 0·0025 g-atom of bivalent vanadium (estimated by methylene blue reduction) per litre. The pH of both solutions was 3·8. The addition of 0·1 ml. of the haemovanadin solution with a micropipette to 14·2 ml. of the cytochrome solution (ratio V: Fe equal to unity) gave a nearly complete reduction of cytochrome, as judged by the slight change in spectrum on subsequent addition of Na2S2O4. The test was then repeated with a stoichiometric ratio V : Fe of one to three. The reduction was much less complete, even after 30 min. We believe that the reaction is represented by V + + + Fe + + + = V + + + + Fe + + rather than by the equation V2O2 +3Fe2O3 = V2O5 + 3Fe2O2, well known to chemists, and that cytochrome c therefore cannot oxidize vanadium beyond the trivalent stage.
We also found that a solution of V2O2 could reduce cytochrome c. The fact that haemovanadin reduces cytochrome c does not imply that this reduction is part of the biological function of the vanadium compound in the animal. Nevertheless, we have found cytochrome c in Phallusia mamillata Cuv. The demonstration in the sperm of the animal is quite easy if sperm suspensions in sea water are warmed to 30° C. or are treated with KCN under the spectroscope. The bands of reduced cytochrome c appear very clearly and resemble those seen in baker’s yeast. Ripe eggs of P. mamillata also contain cytochrome c. Both in the case of sperm and of ripe eggs, we were unable to extract the cytochrome from the living cells, as the rupture of the cells causes the bands to disappear very rapidly. It is interesting to note that a similar instability of cytochrome c in marine animals had been noted by Ball & Meyerhof (1940).
We also tested the blood cells, branchial sac, muscles and stomach of the animal for cytochrome, but we were unable to find it.
The experiments described above are examples of interaction between haemovanadin of Phallusia and cytochrome c from beef heart. We were stimulated by a discussion that one of us (E.B.) had with Dr K. G. Paul of Stockholm to try whether the same interaction could be observed with cytochrome c preparations from Phallusia. Although so far we have been unable to isolate cytochrome c from Phallusia, we have shown that a sperm suspension will show a strong spectrum of reduced cytochrome c after treatment with haemovanadin.
We could find no evidence for a reaction between cyanide and haemovanadin. Several tests, giving easily reproducible results, showed that cyanide did not alter the titration value of haemovanadin against methylene blue; neither did it when haemovanadin was replaced by solutions of V2O2.
We wish to stress with particular emphasis the fact that we do not believe at all that the function of the vanadium compound in the blood of the animal is that of reducing cytochrome c, although we do believe that its physiological function is connected with its reducing properties.
SUMMARY
A method for obtaining stable solutions of haemovanadin from the blood of Phallusia mamillata Cuv. is described. It is essential that all operations be carried out at pH 2·4.
Native haemovanadin reduces cytochrome c both under aerobic and anaerobic conditions. Solutions of V2O2 also reduce cytochrome c. The reduction takes place with a stoichiometric ratio V + +: Fe + + + of one to one.
The sperm and ripe eggs of the animal contain cytochrome c. The pigment was not found in other tissues.
Cyanide does not inhibit the reducing power of haemovanadin towards cytochrome c or methylene blue.
ACKNOWLEDGEMENTS
We wish to express our gratitude to Dr D. Keilin, Cambridge, for his helpful revision of the manuscript and criticism.