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On the nature of vanadium in vanadocyte hemolyzate from ascidians
On the Nature of Vanadium in Vanadocyte Hemolyzate from Ascidians Enzo Boer? and Anders Ehrenberg From
the Medical
Nobel
Institute,
Biochemical
Received November
Department,
Stockholm,
Sweden
12, 1953
INTRODUCTION
The presence of vanadium in blood cells of ascidians was first discovered by Hence in 1911 (1). Some work has been done since that time in order to elucidate the nature of the vanadium present in hemolyzate from these cells. Bielig (private communication) believes it to be trivalent from polarographic measurements. One of us (E. B.) has thought that it was bivalent from oxidative potentiometric measurements (2). Califano and Caselli (3, 4) assumed that the vanadium was present in an organic compound for which they suggested the name “hemovanadin.” In none of these caseshave the conclusions been quite free from objection. Vanadium is a metal for which the determination of the state of valence from its paramagnetic properties is easy. We planned therefore to investigate the paramagnetism of the metal in the hemolyzate from vanadocytes and then to compare some of its properties with the properties of vanadium sulfate of the same valence; the choice of the anion is bound to the fact that the vanadocytes are very rich in sulfate ions (1, 2). MATERIAL Preparations of vanadocyte hemolyzates from Ascidia obliqua Alder and Phallusia mamillata (Cuvier) were studied. The specimens of Ascidia obliqua were taken from Gullmarsfjorden. They were sent in sea water by train in the evening from the Zoological Station at Kristineberg, and arrived at Stockhom the following morning. They were immediately placed in an aquarium filled with freshly prepared artificial sea water at 3-5°C. A stream of air was bubbled continuously 1 Rockefeller Foundation Fellow 1951-1953. Present address : Istituto logia Umana, Universita, Napoli, Italy. 404
di Fisio-
VANADIUM
IN
VANADOCYTE
405
HEMOLYZATE
through the water of the aquarium. By changing the artificial sea water every second day, the animals could be kept alive for more than 1 week. Visually half of the animals were prepared immediately or the day after their arrival, and the others on the 4th~8th days. Before an animal was used, it was checked that its heart was beating. Where it was not, the animal was discarded. In one preparation 40-50 animals were used. The tunica was cut with a small pair of scissors and removed. A cut was made at the heart, and the animal was placed in a clamp hanging in a centrifuge tube and allowed to bleed for some minutes. B stream of commercial nitrogen or carbon dioxide flushed the centrifuge tube the whole time. Contamination with some sea water could not be avoided. Four tubes in parallel were used at a time. When enough blood had been collected, all of it was brought into one tube, flushed with nitrogen, stoppered, and centrifuged for 5 min. at 2500 r.p.m. in a Corda centrifuge. The supernatant and some white blood cells that remained over the green vanadocytes were removed by suction. The vanadocytes were then suspended in fresh artificial sea water (made oxygen-free by bubbling nitrogen) and centrifuged. The washing procedure was repeated three times. The vanadocytes were then suspended in 510 ml. of oxygenfree distilled water and allowed to stand l&15 min. After hemolysis the sample was centrifuged at high speed in a Sorvall centrifuge until the liquid was clear. The sample was transferred into a Thunberg tube and kept, under carbon dioxide. All the steps of the preparation were made at 3-5°C. All samples were analyzed for nitrogen (according to Kjeldahl) sulfur [according to Josephson (5)], vanadium (see under Technique), and dry weight. The pH was measured by means of a glass electrode and a line-operated Cambridge potentiometer. The data of seven preparations are collected in Table I. These preparations were used for spectrophotometric and magnetic determinations. Magnetic determinations were also made on three other preparations: (1) washed vanadocytes from three animals suspended in artificial sea water (oxygenfree) ; (2) and (S), whole blood from each of one large animal kept under an oxygenfree atmosphere for about 3 hr. before bleeding. These samples are named “cell suspension, ” “whole blood 2,” and “whole blood 3,” respectively. They were all analyzed for vanadium and gave the following result,s: 2.81, 4.55, and 1.03 mAl. “Whole blood 2” had a dry weight of 49.52 mg./ml., which upon burning gave 5.20 mg. ash/ml. The preparations of hemolyzates of vanadocytes from PhaUusia mandata (taken in the gulf of Naples) were performed in the same way as described above TABLE Prepn.
I II III IV V VI VII
No.
I
s
V mM
N mg./ml.
Dry weight mg.lml.
PH
?Bg./??d.
2.05 2.34 12.2 7.79 17.9 49.5 14.4
1.371 1.320 1.058 0.994 0.202 0.606 0.406
4.80 2.69 2.53 1.91 1.42 2.55 1.01
31.42 23.87 24.95 20.94 20.94 26.92 19.14
1.19 1.67 1.13 1.75 2.59 2.44 2.23
406
ENZO BOER1 AND ANDERS EHRENBERG
for Ascidia obliqua. In this instance, however, one single animal gave by heart puncture enough blood for a whole preparation. Samples of vanadiumnx were prepared by reducing vanadiumV sulfate in a reduction column with amalgamated zinc at a sulfuric acid concentration of 0.5 M. The vanadium in the outflow was bivalent. The bivalent vanadium was mixed with a proper amount of quinquevalent vanadium to give lOO’% of trivalent vanadium. TECHNIQUE The vanadium content of the samples was determined by reductometric titration with stannous chloride. Before the titration the vanadium must be in the quinquevalent state. For that purpose, the sample was incinerated in hot concentrated sulfuric acid with additions of hydrogen peroxide and nitric acid. Some crystals of potassium chlorate were added at the end in order to obtain complete oxidation to quinquevalent vanadium, and the sample was again heated till dense fumes of sulfuric acid appeared. The sample was then cooled and transferred into the titration vessel and diluted to l-3 mM in vanadium and about 1 IV in sulfuric acid. Addition of solid KaCl proved useful in speeding up the attainment of the electrometric equilibrium. The titration vessel was made of glass with a ground neck (smallest diameter about 40 mm.), to which a ground-brass or a polymethyl methacrylate (Plexiglas) cover fitted. This cover had holes through which electrodes and glass tubes could be inserted and tightened with pieces of rubber. One gas inlet and one outlet, one fork-shaped glass tube carrying two platinum electrodes, one agar-KC1 bridge, and one buret were inserted. The buret were flushed with nitrogen before being filled. The tightness of the system was controlled by keeping the gas outlet under water. Nitrogen was made oxygen-free by pasaing it through a washing flask containing vanadium” sulfate in N sulfuric acid over amalgamated zinc. This oxygen absorber proved excellent; a color change from purple to blue and green indicates exhaustion of the absorber, but the presence of amalgamated zinc makes the absorber efficient for a long time. The purified nitrogen was bubbled through the solution in the titration vessel. The solution was also stirred with a fast magnetic stirrer. The buret was filled with oxygen-free stannous chloride solution, and the vanadium solution was titrated. The potential was measured with a Cambridge potentiometer. A sudden drop in the potential indicated the end point of the titration. The stannous chloride solution was standardized against potassium permanganate in a similar way. The spectrophotometric determinations were made with a Beckman quartz spectrophotometer model DU. Microcells with a l-cm. layer containing 0.3-0.5 ml. of sample were used. All samples were covered with paraffin oil in order to avoid oxidation by air. pH adjustments were made by adding small portions of solid sodium hydrogen carbonate. All extinction values are given in square centimeters per mole of vanadium, using natural logarithms. The magnetic determinations were made in the microapparatus designed by Theorell and Ehrenberg (6).
VANADIUM
Results
of Magnetic
Sample
IN
VANADOCYTE
TABLE II Measurements on Fresh
Gas used during preparation
IIIa IIIb IVa IVb Va Vb VI VII VIII Cell suspension in sea water Whole blood 2
None Kane co2 con co2 co2 Ne N, N, N?
N¶
Whole blood 3
NT
PH
1.13 1.13 l.i3 3.37
2.59 2.59 3.0 2.23 -
-
-
407
HEMOLYZATE
xl,tDoc.
Hemolyzates x 10’ c.g.s., e.m.u.
2,810 2,780 3,340 3,330 3,450 3,440 3,020 3,220 3,370 3,850
(& (A (zk (32 (f (* (xk (zk (h (A
40) 40j 70) 70) 40) 40) 80) 30) 60) 500)
4,100+(1 500) 43,600 3,800 (f750)
(Ascidia
obliqua)
= 2.83 &T Bohrmagmtons
/Jeff
2.56 2.55 2.79 2.79 2.84 2.84 2.66 2.74 2.81 3.0
(z!z (i (A (A (i (i (zk (3~ (3~ (22
3.1-t& 42.9 3.0 (i
0.02) 0.02) 0.03) 0.03) 0.02) 0.02) 0.04) 0.02) 0.03) 0.2) 0.2) 0.3)
A Spinco analytical ultracentrifuge model E was used for the sedimentation studies. All measurements were made at 20°C.
The results of the magnetic determinations on vanadocyte hemolyzates from Bscidia obliqua immediately after their preparation have been collected in Table II. The data have been corrected for the diamagnetism of the organic material and of the salt present in the actual sample in the following way. It was assumedthat all the sulfur (Table I) was present as sulfate ions. The distribution of these sulfate ions between sulfuric acid and potassium sulfate, the only salt assumed to be present, was estimated from the pH of the sample. The correction for sulfate could then be obtained from separate magnetic determinations on properly prepared solutions of sulfuric acid and potassium sulfate. The dry weight (Table I) was corrected for sulfate (sulfuric acid now as ammonium sulfate) and the diamagnetic correction for the rest was estimated from a mean value obtained from measurementson different proteins. (It is noteworthy that all the dry weights corrected as above lie between 13.2 and 14.4 mg./ml., while the total dry weights in Table I vary within the much broader range of 19.9-31.5 mg./ml.) The correction for the sulfate amounted to between 2 and 5 yOand that of the organic material to l-3 yO of the total susceptibility. No correction has been made for the diagmagnetism of vanadium itself.
408
ENZO
BOER1
AND
ANDERS
EHRENBERG
The probable errors introduced in Table II are estimated from probable errors in the measurements and in the diamagnetic corrections. The three last rows of Table II give the results of measurements made directly on the vanadocytes. Here the diamagnetic corrections (IO-20 % of the total susceptibility) are of course much more uncertain than for the hemolyzates. In all three cases a sedimentation of the vanadocytes took place during the measurements. This sedimentation was most rapid for the cell suspension. Only for the preparation “whole blood 2” was it accompanied by a drift in the readings, which might have been caused by a nonuniform sedimentation in the horizontal tube. For perchloric acid solutions of inorganic trivalent vanadium, Freed (7) has found effective Bohr magneton numbers in close agreement with the theoretical values based upon the spin-only theory: 3.88 Bohr magnetons for bivalent vanadium, 2.83 for trivalent, and 1.73 for quadrivalent. If the same theory is applicable to our case, the data show that hemolyzates prepared under exclusion of air contain 100 % (or nearly 100 “r,) trivalent vanadium. If a preparation was made in air (sample III), partial oxidation to quadrivalent vanadium took place. Flushing with commercial nitrogen gave also rise to some oxidation, whereas carbon dioxide gave only trivalent vanadium in the preparation. The pH did not seem to influence the paramagnetic susceptibility to any measurable extent (samples IVu and IV%). On prolonged exposure to air, the paramagnetism of a sample decreased and approached asymptotically the value for quadrivalent vanadium with 1.73 Bohr magnetons. The values obtained on nonhemolyzed vanadocytes in isotonic sea water or in blood are all slightly higher than would be expected if all the vanadium was in the trivalent state. The presence of a small amount of bivalent vanadium should have just this effect. It was attempted to increase the amount of the supposed bivalent vanadium by keeping the animals in anaerobiosis some hours before the blood samples were taken, but no increase in paramagnetism could be detected in this way. However, the present data are not conclusive because of the uncertainty in the diamagnetic correction applied. The spectral properties in the visible region of vanadocyte hemolyzates from As&&u obliqua are presented in Fig. 1. All extinctions are calculated per gram atom vanadium. At pH higher than 2, a sharp peak at 430 rnp is developed, and the solution turns yellow-brown. By trial and error it was found that this transition is best described as a hydrolysis
VANADIUM
5
IN
VANADOCYTE
409
HEMOLYZATE
XIOS
z$ CI e4
3
2
1
FIG. 1. Spectra of vanadocyte hemolyzate from Ascidia obliqua pH: (No.; pH), (1: 2.98), (2: 2.59), (3: 2.44), (4: 2.2.3), (5: 2.16), 1.75). Sulfate-ion concentration = 55 f 25 mM.
of the trivalent written
at different (6: 2.06),
(7:
vanadium involving two hydrogen ions, which can be v-
+ Hz0 s VOf + 2H+
V+++ + 2HzO + V(OH),+
+ 2H+
In Fig. 2 the experimental data have been plotted to represent the logarithmic form of the corresponding mass-action law equation. The ex-
410
EN20
1 FIG.
2. Logarithmic
BOER1
AND
ANDERS
EHRENBERG
2 3 4 PH representation of the spectral change at 430 rnw of the hemolysate from Ascidia obliqua.
trapolated extinction values at 430 rnp, Pv+++ = 0.196 X lob, and p v(OH)l+ = 4.72 X lo6 sq. cm./mole have been used. It is apparent that y pK = pH ao%= 2.51.’ All the hemolysate samplesused had a maximal sulfate-ion concentration of 55 (f25) mM. Vanadium”’ sulfate in sulfuric acid solution exhibits nearly exactly the same spectral properties as the hemolyzates. The only differences found are a somewhat smaller extinction at 300-370 rnp and a slightly more pronounced peak at 610-630 rnp for the inorganic material. Two sets of experiments were performed with varying pH and at, sulfate concentrations of 70 and 750 mM. Both series gave n = 2 and the extrapolated extinction values &+++ = 0.184 X lo6 and /3V(om2+ = 4.72 X lo6 sq. cm./mole at 430 rnp, whereas pHeoS = 2.83 and 3.15, respec-
VANADIUM
IN
VANADOCYTE
HEMOLYZATE
411
tively. The hydrolysis is thus greatly influenced by the sulfate-ion concentration. Our finding that n = 2 for the hydrolysis of V+++ in sulfuric acid milieu is contrary to the conclusion of Furman and Garner (8) that n = 1 in perchloric acid solution. However, their result is based on four experimental points, three of which, as they have shown themselves, are equally well compatible with n = 2. Small contaminations with bi- or quadrivalent vanadium in the hemolyzate could not be detected by the spectrophotometric method, because of the sulfate-ion sensitivity of the hydrolysis constant of trivalent vanadium and the absence of any pronounced absorption peak of either bivalent or quadrivalent vanadium in the spectral range studied. The spectral properties of vanadocyte hemolyzate from Ascidia obliqua that have been presented here did not agree with the results that one of
j-
FIG. 3. Spectra of vanadocyte hemolyzate from ent pH: (No.; pH), (1: 3.50), (9: 3.30), (3: 3.17), 2.13). Sulfate ion concentration = 20 mM.
Phallusia mamillata at. differ(4: 2.64), (6: 2.36), (6: 2.28), (7:
412
ENZO
BOER1
.4ND
ANDERS
EHRENBERG
us (E. B.) had earlier obtained in the case of Phallusia mamillata (2). Repeated experiments on hemolyzates from this species, however, completely confirmed the difference in the absorption properties. As appears in Fig. 3, the extinctions in general are much higher than for Ascidia obliqua (Fig. l), and the maximum at 430 rnp is much less distinct. This could be explained either in terms of complex formation or as a superposition of absorption spectra. The fact that the absorption is greater at all wavelengths speaks perhaps in favor of the latter assumption, while the fact that the tota. extinction depends on the sulfate-ion concentration (see Fig. 4), as the extinction of vanadium’I’ sulfate does, favors the former hypothesis. However, a more detailed investigation is needed to decide this question. The redox potential of the reaction V”’ * VI” + e- was determined on three samples of hemolyzate from Ascidia obliqua (see Fig. 5). The samplehaving the highest pH was exposed to oxidation in air. This autoxidation was followed spectrophotometrically until it had proceeded to
(10
1
5
2
3
4 PH
4. The extinction values at 430 q plotted against pH at different sulfateion concentrations: 0, 20 n&f; +, 100 mM; X, 600 n&f. The broken curve (-; 55 mM) represents the values for hemolyzates from Ascidia obliqua.The other curves derive from Phallusia mamillata. FIG.
VANADIUM
IN
VANADOCYTE
413
HEMOLYZATE
100.
1 FIG. 5. Redox potentials of hemolyeates from Ascidia (2)], and vanadiumIn sulfate
2 for
the
obliqua (+).
3
4pH
transition VIII e VIv + e- at different pH (A), from Phdusia mamillata (0) [see Ref. The curve is calculated.
50 %, when it was interrupted by rapid evacuation of the sample. The sample was divided into three parts, two of which were adjusted to lower pH by addition of sulfuric acid. The redox potentials were measured simultaneously in three small vesselsof the sametype as described under Technique. The potentials were very sluggish and it took several hours before equilibrium seemedto have been established. A sample containing equal parts of vanadiumI” and vanadiumIV sulfate was prepared by mixing equal parts of vanadiumI and vanadiumV sulfate, both in 125 mM sulfuric acid. This was titrated with 3 N NaOH in 125 mM sodium sulfate in a vessel containing a double platinum electrode and one glass electrode so that oxidation-reduction (redox) potential and pH could be measured in parallel. In this case the electrode potential was not at all sluggish. This was partly due to a higher vanadium concentration in comparison with the hemolyzates. The results of these experiments are plotted in Fig. 5. The values obtained earlier by Boeri (2) for the hemolyzates of Phallusia mamillata
414
ENZO
BOER1
AND
ANDERS
EHRENBERG
have also been included in the figure. The full drawn curve is calculated on the assumption that the following reactions are significant.
V(OH)t
V-
* V(OH)F
+ em;
+ 2HzO * V(OH)f
with
+ 2H+;
E = E ,, + B_T ln W(OH)i+l F [V(OH)ZI with Km = [V(OH)tl v-
[H+l” ’
and
V(OH)-
+ Hz0 G V(OH)F
+ H+;
with Km =
W(OH)t+l[H+l DWW+++I
The electrode potential is solved for
W+++l + W(OH):I
= W(OH)+++l+
W(OH)t+l,
which gives
E w s-Eo++(l+
g)-+(‘+g)
Eo is the asymptote of the curve at higher pH values and is thus put equal to 57 mv. K m is: approximated to lo-+ M2 for 0.125 M sulfate from the spectrophotometric experiments. K,, is set equal to 10V2M in order to get the curve to fit in the best way at the lower pH values. The experimental data on inorganic material fits very well to the theoretical curve. The agreement between the curve and the data on hemolyzates from Phallusia mamillata is surprisingly good, as the sulfate-ion content in those experiments was not known. The values obtained with hemolyzates from Ascidia o&qua are a little too high; this may be due in part to some overoxidation to quadrivalent vanadium. The sluggishness of the potential could also have been worse than thought. &&dative potentiometric titrations of bivalent vanadium at pH O-l with permanganate showed that the potential level for equal parts of VII1 and VIv was difficult to obtain in this way. Instead of one broad level, two or three narrower levels were seen. Sometimes these potentials were quite constant for more than 1 hr.; sometimes they drifted more or less slowly. In some cases they did not appear at all. This uncontrolled sluggishnessmay probably be due to an unsatisfactory cleaning of the platinum electrodes. No method of cleaning could be found that always
VANADIUM
IN
VANADOCYTE
HEMOLYZATE
415
gave a good platinum surface. The observed effects fully explain the shape of the titration curve for hemolyzate from Phallusia mamillata that Boeri obtained in his earlier work [Ref. (Z), Fig. 41. This curve illustrates a titration of a mixture of about 80 % V”I and 20 y0 VIv first to 100 $& VIv over two intermediate sluggish levels and then to Vv. Ultracentrifugations were made with four samples of fresh hemolyzate from Ascidia obliqua at 240,000 X g. A low and broad peak with its maximum in the meniscus and a broad tail at the bottom of the cell appeared. The peak had not moved from the meniscus after 5 hr. run. No sedimentation of the color due to trivalent vanadium could be observed. The hemolyzate may thus contain a polydisperse system of molecules with weights mainly less than about 10,000 and more than about 400,000. The vanadium ions are certainly not bound to any molecules with a molecular weight higher than a few thousands. DISCUSSION
The close analogy in the different properties investigated here of vanadocyte hemolyzates and solutions of vanadium1’x sulfate shows that the vanadium in the hemolyzate of vanadocytes is trivalent and very probably present as vanadium ions. The complexing, if any, must be very weak in nature. This point of view is in line with that of Lybing (9), who has also studied the spectral properties of vanadocyte hemolyzates of Ascidia obliqua. It is also well compatible with the work of Califano and Caselli (3) who demonstrated that vanadocyte hemolyzates from Phallusia mamillata contain a protein that can be released by precipitation with a strong acid, shaking with chloroform, or ultrafiltration. They concluded that the protein is either independent or weakly bound to the vanadium. Califano and Caselli have also studied the spectral properties of vanadocyte hemolyzate from Phallusia mamillata in the ultraviolet region (4). They state that the absorption band observed there is an expression of an organic vanadium compound as it is not simply referable to the vanadium. This statement must be regarded as a mere assumption as they do not discuss the possibility that the observed spectrum is the sum of that of vanadium and that of one or several organic compounds. Califano and Caselli also mention that the hemolyzate from Phallusia mamillata gives in ultraviolet light a fluorescence at about 470 rnp (4).
416
ENZO BOER1 AND ANDERS EHRENBERG
We have controlled and seen the same fluorescence in the hemolyzate from Asciclia obliqua, where it is fainter. As vanadium sulfate solutions of any state of valence do not give any fluorescence, the effect is certainly due to some organic material. The properties of the vanadium in vanadocyte hemolyzates do not give any conclusive information about the properties of vanadium in the much more condensed system inside the vanadocyte. The vanadium in the cell may well to some extent be bound to the cell membrane or to some protein. The state of valence is probably the same as in the hemolyzate. Further investigation will show whether the peculiar absorption of hemolyzates from Phallusia mamilZata are due to any complex formation. ACKNOWLEDGMENTS The authors wish to thank Professor H. Theorell for his kind interest during the course of the work, Dr. G. Gustafsson at Kristineberg for supplying the Ascidia obliqua animals, and the Svenska Vetenskapsakademien for the hospitality at Kristineberg. Thanks are also due to the Rockefeller Foundation for a fellowship (to E. B.) and to Magnus Bergvalls Stiftelse for a grant (to A. E.). SUMMARY
It has been shown by magnetic, spectrophotometric, reductometric, and ultracentrifugation experiments that the vanadium in vanadocyte hemolyzates from Ascidia obliqua and Phallusia mamillata is present as trivalent vanadium sulfate. The absorption peak at 430 rnp is due to the hydrolysed ion V(OH)s+ (or VO+). The hydrolysis constant is found by spectrophotometric measurements to be dependent on the sulfate-ion concentration. REFERENCES 1. 2. 3. 4. 5. 6. 7, 8. 9.
HENZE, M.,Z. physiol. Chem.72,494 (1911). BOERI, E., Arch. Biochem. and Biophys. 3’7, 449 (1952). CALIFANO, L., AND CASELLI, P., Pubbl. slaz. zool. Napoli 21, 235 (1947). CALIFANO, L., AND CASELLI, P., Pubbl. staz. zool. Napoli 23, 1 (1959). JOSEPHSON, B., Analyst 64, 181 (1939). THEORELL, H., AND EHRENBERG, A., A&iv Fysik 3, 299 (1951). FREED, S., J. Am. Chem. Sot. 49, 2456 (1927). FURMAN, S. C., AND GARNER, C. S., J. Am. Chem. Sot. 73,1785 (1950). LYBING, S., Arkiv Kemi 6, No. 21 (1953).
the Medical
Nobel
Institute,
Biochemical
Received November
Department,
Stockholm,
Sweden
12, 1953
INTRODUCTION
The presence of vanadium in blood cells of ascidians was first discovered by Hence in 1911 (1). Some work has been done since that time in order to elucidate the nature of the vanadium present in hemolyzate from these cells. Bielig (private communication) believes it to be trivalent from polarographic measurements. One of us (E. B.) has thought that it was bivalent from oxidative potentiometric measurements (2). Califano and Caselli (3, 4) assumed that the vanadium was present in an organic compound for which they suggested the name “hemovanadin.” In none of these caseshave the conclusions been quite free from objection. Vanadium is a metal for which the determination of the state of valence from its paramagnetic properties is easy. We planned therefore to investigate the paramagnetism of the metal in the hemolyzate from vanadocytes and then to compare some of its properties with the properties of vanadium sulfate of the same valence; the choice of the anion is bound to the fact that the vanadocytes are very rich in sulfate ions (1, 2). MATERIAL Preparations of vanadocyte hemolyzates from Ascidia obliqua Alder and Phallusia mamillata (Cuvier) were studied. The specimens of Ascidia obliqua were taken from Gullmarsfjorden. They were sent in sea water by train in the evening from the Zoological Station at Kristineberg, and arrived at Stockhom the following morning. They were immediately placed in an aquarium filled with freshly prepared artificial sea water at 3-5°C. A stream of air was bubbled continuously 1 Rockefeller Foundation Fellow 1951-1953. Present address : Istituto logia Umana, Universita, Napoli, Italy. 404
di Fisio-
VANADIUM
IN
VANADOCYTE
405
HEMOLYZATE
through the water of the aquarium. By changing the artificial sea water every second day, the animals could be kept alive for more than 1 week. Visually half of the animals were prepared immediately or the day after their arrival, and the others on the 4th~8th days. Before an animal was used, it was checked that its heart was beating. Where it was not, the animal was discarded. In one preparation 40-50 animals were used. The tunica was cut with a small pair of scissors and removed. A cut was made at the heart, and the animal was placed in a clamp hanging in a centrifuge tube and allowed to bleed for some minutes. B stream of commercial nitrogen or carbon dioxide flushed the centrifuge tube the whole time. Contamination with some sea water could not be avoided. Four tubes in parallel were used at a time. When enough blood had been collected, all of it was brought into one tube, flushed with nitrogen, stoppered, and centrifuged for 5 min. at 2500 r.p.m. in a Corda centrifuge. The supernatant and some white blood cells that remained over the green vanadocytes were removed by suction. The vanadocytes were then suspended in fresh artificial sea water (made oxygen-free by bubbling nitrogen) and centrifuged. The washing procedure was repeated three times. The vanadocytes were then suspended in 510 ml. of oxygenfree distilled water and allowed to stand l&15 min. After hemolysis the sample was centrifuged at high speed in a Sorvall centrifuge until the liquid was clear. The sample was transferred into a Thunberg tube and kept, under carbon dioxide. All the steps of the preparation were made at 3-5°C. All samples were analyzed for nitrogen (according to Kjeldahl) sulfur [according to Josephson (5)], vanadium (see under Technique), and dry weight. The pH was measured by means of a glass electrode and a line-operated Cambridge potentiometer. The data of seven preparations are collected in Table I. These preparations were used for spectrophotometric and magnetic determinations. Magnetic determinations were also made on three other preparations: (1) washed vanadocytes from three animals suspended in artificial sea water (oxygenfree) ; (2) and (S), whole blood from each of one large animal kept under an oxygenfree atmosphere for about 3 hr. before bleeding. These samples are named “cell suspension, ” “whole blood 2,” and “whole blood 3,” respectively. They were all analyzed for vanadium and gave the following result,s: 2.81, 4.55, and 1.03 mAl. “Whole blood 2” had a dry weight of 49.52 mg./ml., which upon burning gave 5.20 mg. ash/ml. The preparations of hemolyzates of vanadocytes from PhaUusia mandata (taken in the gulf of Naples) were performed in the same way as described above TABLE Prepn.
I II III IV V VI VII
No.
I
s
V mM
N mg./ml.
Dry weight mg.lml.
PH
?Bg./??d.
2.05 2.34 12.2 7.79 17.9 49.5 14.4
1.371 1.320 1.058 0.994 0.202 0.606 0.406
4.80 2.69 2.53 1.91 1.42 2.55 1.01
31.42 23.87 24.95 20.94 20.94 26.92 19.14
1.19 1.67 1.13 1.75 2.59 2.44 2.23
406
ENZO BOER1 AND ANDERS EHRENBERG
for Ascidia obliqua. In this instance, however, one single animal gave by heart puncture enough blood for a whole preparation. Samples of vanadiumnx were prepared by reducing vanadiumV sulfate in a reduction column with amalgamated zinc at a sulfuric acid concentration of 0.5 M. The vanadium in the outflow was bivalent. The bivalent vanadium was mixed with a proper amount of quinquevalent vanadium to give lOO’% of trivalent vanadium. TECHNIQUE The vanadium content of the samples was determined by reductometric titration with stannous chloride. Before the titration the vanadium must be in the quinquevalent state. For that purpose, the sample was incinerated in hot concentrated sulfuric acid with additions of hydrogen peroxide and nitric acid. Some crystals of potassium chlorate were added at the end in order to obtain complete oxidation to quinquevalent vanadium, and the sample was again heated till dense fumes of sulfuric acid appeared. The sample was then cooled and transferred into the titration vessel and diluted to l-3 mM in vanadium and about 1 IV in sulfuric acid. Addition of solid KaCl proved useful in speeding up the attainment of the electrometric equilibrium. The titration vessel was made of glass with a ground neck (smallest diameter about 40 mm.), to which a ground-brass or a polymethyl methacrylate (Plexiglas) cover fitted. This cover had holes through which electrodes and glass tubes could be inserted and tightened with pieces of rubber. One gas inlet and one outlet, one fork-shaped glass tube carrying two platinum electrodes, one agar-KC1 bridge, and one buret were inserted. The buret were flushed with nitrogen before being filled. The tightness of the system was controlled by keeping the gas outlet under water. Nitrogen was made oxygen-free by pasaing it through a washing flask containing vanadium” sulfate in N sulfuric acid over amalgamated zinc. This oxygen absorber proved excellent; a color change from purple to blue and green indicates exhaustion of the absorber, but the presence of amalgamated zinc makes the absorber efficient for a long time. The purified nitrogen was bubbled through the solution in the titration vessel. The solution was also stirred with a fast magnetic stirrer. The buret was filled with oxygen-free stannous chloride solution, and the vanadium solution was titrated. The potential was measured with a Cambridge potentiometer. A sudden drop in the potential indicated the end point of the titration. The stannous chloride solution was standardized against potassium permanganate in a similar way. The spectrophotometric determinations were made with a Beckman quartz spectrophotometer model DU. Microcells with a l-cm. layer containing 0.3-0.5 ml. of sample were used. All samples were covered with paraffin oil in order to avoid oxidation by air. pH adjustments were made by adding small portions of solid sodium hydrogen carbonate. All extinction values are given in square centimeters per mole of vanadium, using natural logarithms. The magnetic determinations were made in the microapparatus designed by Theorell and Ehrenberg (6).
VANADIUM
Results
of Magnetic
Sample
IN
VANADOCYTE
TABLE II Measurements on Fresh
Gas used during preparation
IIIa IIIb IVa IVb Va Vb VI VII VIII Cell suspension in sea water Whole blood 2
None Kane co2 con co2 co2 Ne N, N, N?
N¶
Whole blood 3
NT
PH
1.13 1.13 l.i3 3.37
2.59 2.59 3.0 2.23 -
-
-
407
HEMOLYZATE
xl,tDoc.
Hemolyzates x 10’ c.g.s., e.m.u.
2,810 2,780 3,340 3,330 3,450 3,440 3,020 3,220 3,370 3,850
(& (A (zk (32 (f (* (xk (zk (h (A
40) 40j 70) 70) 40) 40) 80) 30) 60) 500)
4,100+(1 500) 43,600 3,800 (f750)
(Ascidia
obliqua)
= 2.83 &T Bohrmagmtons
/Jeff
2.56 2.55 2.79 2.79 2.84 2.84 2.66 2.74 2.81 3.0
(z!z (i (A (A (i (i (zk (3~ (3~ (22
3.1-t& 42.9 3.0 (i
0.02) 0.02) 0.03) 0.03) 0.02) 0.02) 0.04) 0.02) 0.03) 0.2) 0.2) 0.3)
A Spinco analytical ultracentrifuge model E was used for the sedimentation studies. All measurements were made at 20°C.
The results of the magnetic determinations on vanadocyte hemolyzates from Bscidia obliqua immediately after their preparation have been collected in Table II. The data have been corrected for the diamagnetism of the organic material and of the salt present in the actual sample in the following way. It was assumedthat all the sulfur (Table I) was present as sulfate ions. The distribution of these sulfate ions between sulfuric acid and potassium sulfate, the only salt assumed to be present, was estimated from the pH of the sample. The correction for sulfate could then be obtained from separate magnetic determinations on properly prepared solutions of sulfuric acid and potassium sulfate. The dry weight (Table I) was corrected for sulfate (sulfuric acid now as ammonium sulfate) and the diamagnetic correction for the rest was estimated from a mean value obtained from measurementson different proteins. (It is noteworthy that all the dry weights corrected as above lie between 13.2 and 14.4 mg./ml., while the total dry weights in Table I vary within the much broader range of 19.9-31.5 mg./ml.) The correction for the sulfate amounted to between 2 and 5 yOand that of the organic material to l-3 yO of the total susceptibility. No correction has been made for the diagmagnetism of vanadium itself.
408
ENZO
BOER1
AND
ANDERS
EHRENBERG
The probable errors introduced in Table II are estimated from probable errors in the measurements and in the diamagnetic corrections. The three last rows of Table II give the results of measurements made directly on the vanadocytes. Here the diamagnetic corrections (IO-20 % of the total susceptibility) are of course much more uncertain than for the hemolyzates. In all three cases a sedimentation of the vanadocytes took place during the measurements. This sedimentation was most rapid for the cell suspension. Only for the preparation “whole blood 2” was it accompanied by a drift in the readings, which might have been caused by a nonuniform sedimentation in the horizontal tube. For perchloric acid solutions of inorganic trivalent vanadium, Freed (7) has found effective Bohr magneton numbers in close agreement with the theoretical values based upon the spin-only theory: 3.88 Bohr magnetons for bivalent vanadium, 2.83 for trivalent, and 1.73 for quadrivalent. If the same theory is applicable to our case, the data show that hemolyzates prepared under exclusion of air contain 100 % (or nearly 100 “r,) trivalent vanadium. If a preparation was made in air (sample III), partial oxidation to quadrivalent vanadium took place. Flushing with commercial nitrogen gave also rise to some oxidation, whereas carbon dioxide gave only trivalent vanadium in the preparation. The pH did not seem to influence the paramagnetic susceptibility to any measurable extent (samples IVu and IV%). On prolonged exposure to air, the paramagnetism of a sample decreased and approached asymptotically the value for quadrivalent vanadium with 1.73 Bohr magnetons. The values obtained on nonhemolyzed vanadocytes in isotonic sea water or in blood are all slightly higher than would be expected if all the vanadium was in the trivalent state. The presence of a small amount of bivalent vanadium should have just this effect. It was attempted to increase the amount of the supposed bivalent vanadium by keeping the animals in anaerobiosis some hours before the blood samples were taken, but no increase in paramagnetism could be detected in this way. However, the present data are not conclusive because of the uncertainty in the diamagnetic correction applied. The spectral properties in the visible region of vanadocyte hemolyzates from As&&u obliqua are presented in Fig. 1. All extinctions are calculated per gram atom vanadium. At pH higher than 2, a sharp peak at 430 rnp is developed, and the solution turns yellow-brown. By trial and error it was found that this transition is best described as a hydrolysis
VANADIUM
5
IN
VANADOCYTE
409
HEMOLYZATE
XIOS
z$ CI e4
3
2
1
FIG. 1. Spectra of vanadocyte hemolyzate from Ascidia obliqua pH: (No.; pH), (1: 2.98), (2: 2.59), (3: 2.44), (4: 2.2.3), (5: 2.16), 1.75). Sulfate-ion concentration = 55 f 25 mM.
of the trivalent written
at different (6: 2.06),
(7:
vanadium involving two hydrogen ions, which can be v-
+ Hz0 s VOf + 2H+
V+++ + 2HzO + V(OH),+
+ 2H+
In Fig. 2 the experimental data have been plotted to represent the logarithmic form of the corresponding mass-action law equation. The ex-
410
EN20
1 FIG.
2. Logarithmic
BOER1
AND
ANDERS
EHRENBERG
2 3 4 PH representation of the spectral change at 430 rnw of the hemolysate from Ascidia obliqua.
trapolated extinction values at 430 rnp, Pv+++ = 0.196 X lob, and p v(OH)l+ = 4.72 X lo6 sq. cm./mole have been used. It is apparent that y pK = pH ao%= 2.51.’ All the hemolysate samplesused had a maximal sulfate-ion concentration of 55 (f25) mM. Vanadium”’ sulfate in sulfuric acid solution exhibits nearly exactly the same spectral properties as the hemolyzates. The only differences found are a somewhat smaller extinction at 300-370 rnp and a slightly more pronounced peak at 610-630 rnp for the inorganic material. Two sets of experiments were performed with varying pH and at, sulfate concentrations of 70 and 750 mM. Both series gave n = 2 and the extrapolated extinction values &+++ = 0.184 X lo6 and /3V(om2+ = 4.72 X lo6 sq. cm./mole at 430 rnp, whereas pHeoS = 2.83 and 3.15, respec-
VANADIUM
IN
VANADOCYTE
HEMOLYZATE
411
tively. The hydrolysis is thus greatly influenced by the sulfate-ion concentration. Our finding that n = 2 for the hydrolysis of V+++ in sulfuric acid milieu is contrary to the conclusion of Furman and Garner (8) that n = 1 in perchloric acid solution. However, their result is based on four experimental points, three of which, as they have shown themselves, are equally well compatible with n = 2. Small contaminations with bi- or quadrivalent vanadium in the hemolyzate could not be detected by the spectrophotometric method, because of the sulfate-ion sensitivity of the hydrolysis constant of trivalent vanadium and the absence of any pronounced absorption peak of either bivalent or quadrivalent vanadium in the spectral range studied. The spectral properties of vanadocyte hemolyzate from Ascidia obliqua that have been presented here did not agree with the results that one of
j-
FIG. 3. Spectra of vanadocyte hemolyzate from ent pH: (No.; pH), (1: 3.50), (9: 3.30), (3: 3.17), 2.13). Sulfate ion concentration = 20 mM.
Phallusia mamillata at. differ(4: 2.64), (6: 2.36), (6: 2.28), (7:
412
ENZO
BOER1
.4ND
ANDERS
EHRENBERG
us (E. B.) had earlier obtained in the case of Phallusia mamillata (2). Repeated experiments on hemolyzates from this species, however, completely confirmed the difference in the absorption properties. As appears in Fig. 3, the extinctions in general are much higher than for Ascidia obliqua (Fig. l), and the maximum at 430 rnp is much less distinct. This could be explained either in terms of complex formation or as a superposition of absorption spectra. The fact that the absorption is greater at all wavelengths speaks perhaps in favor of the latter assumption, while the fact that the tota. extinction depends on the sulfate-ion concentration (see Fig. 4), as the extinction of vanadium’I’ sulfate does, favors the former hypothesis. However, a more detailed investigation is needed to decide this question. The redox potential of the reaction V”’ * VI” + e- was determined on three samples of hemolyzate from Ascidia obliqua (see Fig. 5). The samplehaving the highest pH was exposed to oxidation in air. This autoxidation was followed spectrophotometrically until it had proceeded to
(10
1
5
2
3
4 PH
4. The extinction values at 430 q plotted against pH at different sulfateion concentrations: 0, 20 n&f; +, 100 mM; X, 600 n&f. The broken curve (-; 55 mM) represents the values for hemolyzates from Ascidia obliqua.The other curves derive from Phallusia mamillata. FIG.
VANADIUM
IN
VANADOCYTE
413
HEMOLYZATE
100.
1 FIG. 5. Redox potentials of hemolyeates from Ascidia (2)], and vanadiumIn sulfate
2 for
the
obliqua (+).
3
4pH
transition VIII e VIv + e- at different pH (A), from Phdusia mamillata (0) [see Ref. The curve is calculated.
50 %, when it was interrupted by rapid evacuation of the sample. The sample was divided into three parts, two of which were adjusted to lower pH by addition of sulfuric acid. The redox potentials were measured simultaneously in three small vesselsof the sametype as described under Technique. The potentials were very sluggish and it took several hours before equilibrium seemedto have been established. A sample containing equal parts of vanadiumI” and vanadiumIV sulfate was prepared by mixing equal parts of vanadiumI and vanadiumV sulfate, both in 125 mM sulfuric acid. This was titrated with 3 N NaOH in 125 mM sodium sulfate in a vessel containing a double platinum electrode and one glass electrode so that oxidation-reduction (redox) potential and pH could be measured in parallel. In this case the electrode potential was not at all sluggish. This was partly due to a higher vanadium concentration in comparison with the hemolyzates. The results of these experiments are plotted in Fig. 5. The values obtained earlier by Boeri (2) for the hemolyzates of Phallusia mamillata
414
ENZO
BOER1
AND
ANDERS
EHRENBERG
have also been included in the figure. The full drawn curve is calculated on the assumption that the following reactions are significant.
V(OH)t
V-
* V(OH)F
+ em;
+ 2HzO * V(OH)f
with
+ 2H+;
E = E ,, + B_T ln W(OH)i+l F [V(OH)ZI with Km = [V(OH)tl v-
[H+l” ’
and
V(OH)-
+ Hz0 G V(OH)F
+ H+;
with Km =
W(OH)t+l[H+l DWW+++I
The electrode potential is solved for
W+++l + W(OH):I
= W(OH)+++l+
W(OH)t+l,
which gives
E w s-Eo++(l+
g)-+(‘+g)
Eo is the asymptote of the curve at higher pH values and is thus put equal to 57 mv. K m is: approximated to lo-+ M2 for 0.125 M sulfate from the spectrophotometric experiments. K,, is set equal to 10V2M in order to get the curve to fit in the best way at the lower pH values. The experimental data on inorganic material fits very well to the theoretical curve. The agreement between the curve and the data on hemolyzates from Phallusia mamillata is surprisingly good, as the sulfate-ion content in those experiments was not known. The values obtained with hemolyzates from Ascidia o&qua are a little too high; this may be due in part to some overoxidation to quadrivalent vanadium. The sluggishness of the potential could also have been worse than thought. &&dative potentiometric titrations of bivalent vanadium at pH O-l with permanganate showed that the potential level for equal parts of VII1 and VIv was difficult to obtain in this way. Instead of one broad level, two or three narrower levels were seen. Sometimes these potentials were quite constant for more than 1 hr.; sometimes they drifted more or less slowly. In some cases they did not appear at all. This uncontrolled sluggishnessmay probably be due to an unsatisfactory cleaning of the platinum electrodes. No method of cleaning could be found that always
VANADIUM
IN
VANADOCYTE
HEMOLYZATE
415
gave a good platinum surface. The observed effects fully explain the shape of the titration curve for hemolyzate from Phallusia mamillata that Boeri obtained in his earlier work [Ref. (Z), Fig. 41. This curve illustrates a titration of a mixture of about 80 % V”I and 20 y0 VIv first to 100 $& VIv over two intermediate sluggish levels and then to Vv. Ultracentrifugations were made with four samples of fresh hemolyzate from Ascidia obliqua at 240,000 X g. A low and broad peak with its maximum in the meniscus and a broad tail at the bottom of the cell appeared. The peak had not moved from the meniscus after 5 hr. run. No sedimentation of the color due to trivalent vanadium could be observed. The hemolyzate may thus contain a polydisperse system of molecules with weights mainly less than about 10,000 and more than about 400,000. The vanadium ions are certainly not bound to any molecules with a molecular weight higher than a few thousands. DISCUSSION
The close analogy in the different properties investigated here of vanadocyte hemolyzates and solutions of vanadium1’x sulfate shows that the vanadium in the hemolyzate of vanadocytes is trivalent and very probably present as vanadium ions. The complexing, if any, must be very weak in nature. This point of view is in line with that of Lybing (9), who has also studied the spectral properties of vanadocyte hemolyzates of Ascidia obliqua. It is also well compatible with the work of Califano and Caselli (3) who demonstrated that vanadocyte hemolyzates from Phallusia mamillata contain a protein that can be released by precipitation with a strong acid, shaking with chloroform, or ultrafiltration. They concluded that the protein is either independent or weakly bound to the vanadium. Califano and Caselli have also studied the spectral properties of vanadocyte hemolyzate from Phallusia mamillata in the ultraviolet region (4). They state that the absorption band observed there is an expression of an organic vanadium compound as it is not simply referable to the vanadium. This statement must be regarded as a mere assumption as they do not discuss the possibility that the observed spectrum is the sum of that of vanadium and that of one or several organic compounds. Califano and Caselli also mention that the hemolyzate from Phallusia mamillata gives in ultraviolet light a fluorescence at about 470 rnp (4).
416
ENZO BOER1 AND ANDERS EHRENBERG
We have controlled and seen the same fluorescence in the hemolyzate from Asciclia obliqua, where it is fainter. As vanadium sulfate solutions of any state of valence do not give any fluorescence, the effect is certainly due to some organic material. The properties of the vanadium in vanadocyte hemolyzates do not give any conclusive information about the properties of vanadium in the much more condensed system inside the vanadocyte. The vanadium in the cell may well to some extent be bound to the cell membrane or to some protein. The state of valence is probably the same as in the hemolyzate. Further investigation will show whether the peculiar absorption of hemolyzates from Phallusia mamilZata are due to any complex formation. ACKNOWLEDGMENTS The authors wish to thank Professor H. Theorell for his kind interest during the course of the work, Dr. G. Gustafsson at Kristineberg for supplying the Ascidia obliqua animals, and the Svenska Vetenskapsakademien for the hospitality at Kristineberg. Thanks are also due to the Rockefeller Foundation for a fellowship (to E. B.) and to Magnus Bergvalls Stiftelse for a grant (to A. E.). SUMMARY
It has been shown by magnetic, spectrophotometric, reductometric, and ultracentrifugation experiments that the vanadium in vanadocyte hemolyzates from Ascidia obliqua and Phallusia mamillata is present as trivalent vanadium sulfate. The absorption peak at 430 rnp is due to the hydrolysed ion V(OH)s+ (or VO+). The hydrolysis constant is found by spectrophotometric measurements to be dependent on the sulfate-ion concentration. REFERENCES 1. 2. 3. 4. 5. 6. 7, 8. 9.
HENZE, M.,Z. physiol. Chem.72,494 (1911). BOERI, E., Arch. Biochem. and Biophys. 3’7, 449 (1952). CALIFANO, L., AND CASELLI, P., Pubbl. slaz. zool. Napoli 21, 235 (1947). CALIFANO, L., AND CASELLI, P., Pubbl. staz. zool. Napoli 23, 1 (1959). JOSEPHSON, B., Analyst 64, 181 (1939). THEORELL, H., AND EHRENBERG, A., A&iv Fysik 3, 299 (1951). FREED, S., J. Am. Chem. Sot. 49, 2456 (1927). FURMAN, S. C., AND GARNER, C. S., J. Am. Chem. Sot. 73,1785 (1950). LYBING, S., Arkiv Kemi 6, No. 21 (1953).