Buffers in the blood of the snail,helix pomatia L.

Comp. Biochem. Physiol., 1969, Vol. 29, pp. 919 to 930. Pergamon Press. Printed in Great Britain

BUFFERS IN THE BLOOD OF THE SNAIL, H E L I X P O M A T I A L. R. F. B U R T O N Institute of Physiology, University of Glasgow, Glasgow W.2 (Received 21 October 1968)

Abstract--1. Blood from the haemocoels of active snails had an average pH of 7-76. In aestivating snails, it tended to contain less bicarbonate and to have a higher pCO2, so being more acid (mean: pH 7"51). 2. Blood from the heart was 0.12 pH units more alkaline. 3. The pH of maximum buffering by haemocyanin is lowered in the presence of salts, particularly of calcium or magnesium. 4. In vivo, oxyhaemocyanin has a maximum buffer index of 0"17 m-equiv./ pH per g at pH 7.5, contributing little to total buffering but much to carbon dioxide transport. 5. There is a small positive "Haldane effect" that is reversed below pH 7"7. INTRODUCTION THE pH of the blood of Helix pomatia L. varies considerably. Spoek et al. (1964) recorded values of 7.62-8.08 for samples drawn from the heart, the higher values being obtained at lower temperatures. Vorwohl (1961) gave a range of 7.84-8.16 and Raffy & Fischer (1935) one of 7.8-7.95. Sorokina (1965) found that the pH was lowest, about 7.5, in the summer but closer to 8"0 in the winter. The two main buffers in the blood are bicarbonate and the respiratory protein, haemocyanin. Carbon dioxide dissociation curves reflecting this have been published by Nitzescu & Cosma (1927), Wolvekamp & Kruyt (19A.A. 47) and Spoek et al. (1964) and actual determinations of the carbon dioxide content of the blood accord with these. Thus, Duval (1930) found 44-61 ml (N.T.P.)/100 ml and Rally & Fischer (1935), 32.7-75 ml/100 ml. This carbon dioxide must be present mainly as bicarbonate, with some in simple solution and as carbonate, since little, if any, carbamate is formed (Wolvekamp & Kruyt, 1944--47). Thus, the concentration of bicarbonate in the blood would be about 15-34 mM/1. Sorokina & Zelenskaya (1967), however, found only 5.71 + 0.36 mM/1. The concentration of haemocyanin depends on the absolute amount present and on the degree of hydration of the snail, the range being about 7-90 g/kg water (Burton, 1965a, b). Nitzescu & Cosma (1927) did not consider it as playing the main role in the acid-base equilibrium, while Sorokina & Zelenskaya (1967) found, on the other hand, that precipitation of proteins greatly reduced the buffer capacity of the blood. (Whether the bicarbonate would also have been influenced by the 919

920

R . F . BURTON

t r e a t m e n t is not clear.) T e r r o u x (1936) regarded haemocyanin as the major buffer and showed that it had, after dialysis against distilled water, a peak of buffering power at about p H 8.2. ( T h e buffering power also increased below p H 5 and above p H 8.5 b u t this p a p e r is concerned only with physiologically important buffer groups.) I n titrations of natural sera, however, buffering was greatest at about p H 7.5 and the buffer index was over three times as high for a given copper concentration as in dialysed solutions. I t is not clear how the results obtained with sera were affected b y the presence of bicarbonate. Roche (1932) found that purified haemocyanin had a m a x i m u m buffering power at p H 8"6, but also a lower peak which varied with the state of oxygenation of the protein, occurring at p H 7.0-7.1 in the presence of oxygen and at p H 7.6-7.7 in its absence. N o such " H a l d a n e effect" could be detected b y W o l v e k a m p & K r u y t (1944-47) although there are complex Bohr effects. T h i s p a p e r reports investigations on the p H , pCO~ and concentrations of bicarbonate, in vivo, and on the influence of p H , salts and oxygen tension on the buffering properties of haemocyanin. T h e roles of bicarbonate and haemocyanin in the separate buffer functions of carbon dioxide carriage and stabilizing p H are then discussed. MATERIALS AND M E T H O D S Buffer index curves and "Haldane effects" Samples of blood, pooled from several snails, were centrifuged to remove cells and other particles. Subsequent treatment, prior to titration, varied: (1) The blood was acidified to pH 5-6 with 0"5 N HCI in the titration vessel and the carbon dioxide removed by equilibration with oxygen. (2) The blood was diluted with a similar volume of saline containing 60 mM/l of NaCI, 10 mM/l of CaC12 and 14 raM/1 of MgC12 and was centrifuged at 5°C and at 8 x 107 cm/ secs (average) to throw down the haemocyanin. The latter was then redispersed in more of the same saline. Solutions used in the investigation of the Haldane effect were prepared in this way. (3) Haemocyanin concentrated by centrifugation, as in (2), was redispersed in distilled water, or in 0'04 or 0"1 M NaC1, before being dialysed against the same. Concentrations of haemocyanin were measured by weighing samples before and after drying at 105°C. Titrations were carried out at room temperature (20-25°C) on 5-ml aliquots in a cylindrical glass vessel of capacity 20 ml. The latter was covered with "Parafilm" through which passed glass and reference electrodes (Radiometer), the tip of a microburette containing acid or alkali and a tube for the delivery of a continuous flow of oxygen. Samples were freed of carbon dioxide by equilibration with oxygen before being titrated. They were stirred magnetically except when readings of pH were being taken for solutions of low ionic strength. During titrations, 0"5 N NaOH or 0"5 N HCI were added in steps usually of 0"001 m-equiv, between readings of pH. The buffer index, defined as d (base)/ d (pH), was calculated from the change in pH resulting from the addition of 0.002 m-equiv. of acid or alkali. For the investigation of the Haldane effects, solutions of haemocyanin in saline were prepared as described above. About 4 ml were placed in a glass vessel similar to that used for the titrations, but taller, and the electrodes inserted through a "Parafilm" cover. The solution was stirred magnetically and equilibrated alternately with nitrogen and with 5' 1% oxygen in nitrogen which bubbled through it. These gases were passed through solutions

BUFFERS I N THE BLOOD OF THE SNAIL

921

of barium hydroxide to remove traces of carbon dioxide. The p H of the solution of haemocyanin was adjusted with HCI and NaOH. p H and carbon dioxide tension, in vivo

Blood samples of about 50/~1 were collected in capillary tubes, as far as possible anaerobically, from just under the thin membrane covering the digestive gland. The pH was measured at 20°C by means of a Radiometer p H meter 27 with micro-electrode unit. The snails were also at about this temperature. Usually three samples were collected from each snail and the results averaged. Further blood was collected after the anaerobic samples and used to determine carbon dioxide tension (pCOs) by the method of Astrup (1956) and Siggaard Andersen et al. (1960), assuming a linear relation between pH and log (pCOt) and making use of the measured value of the pH in vivo. Calculation of concentrations of bicarbonate and carbonate

Concentrations of bicarbonate, in raM/l, were calculated from the relation: log[HCO3'] = pH + log(pCO2) - Q, where pCO~ is in mm Hg. The constant Q depends on the apparent first dissociation constant of carbonic acid and on the solubility of carbon dioxide and, therefore, also on temperature and ionic strength. Since published values for apparent first dissociation constants are not in agreement, Q was determined empirically for solutions of NaC1 and NaHCO3. As indicated by the data of Burton (1965b), the ionic strength (/0 of the blood under normal conditions is variable, but a value of 0"15 was used in the calculation of bicarbonate levels in vivo. The corresponding value of Q at 20°C was taken to be 7"483. Errors in the calculation of bicarbonate levels due to deviations of ionic strength from 0"15 would be less than 5 per cent. Data of Burton (1968) suggest that the ionic strength of the blood from the fully hydrated snails used for titrations with carbon dioxide would have been about 0'12 and, for these, Q at 20°C was taken as 7"500. Carbonate concentrations were calculated from the relation: log[COa"] = pH + log[HCOa'] - p K ( . p K ( was taken to be (10"255 -1"1~/~) at 20°C (Hastings & Sendroy, 1925; Kauko & Airola, 1937). RESULTS Buffer index curves were determined for four samples of bicarbonate-free oxygenated blood originally containing 19-29 g of solutes/kg. I n each case, m a x i m u m buffering occurred close to p H 7.5 and the curves corresponded w h e n drawn with their peaks adjusted to the same height. T h e m e a n curve, corresponding to blood containing 24 g of sohites/kg, is shown in Fig. 1 (curve 1). Part of the buffer capacity of bicarbonate-free blood is not due to haemocyanin and the contribution of other substances is shown b y curve 2 in Fig. 1. T h i s represents the m e a n of three curves obtained b y the titration of blood freed of haemocyanin b y centrifugation and of bicarbonate b y acidification u n d e r oxygen. T h e s e samples contained 6.5-7.1 g of solutes/kg. Allowing for the contribution of these substances to the buffer index and solute content of blood, the m a x i m u m buffer index of the haemocyanin in the blood was calculated to be 0.17 m - e q u i v . / p H per g. T h e third curve in Fig. 1 is based on titrations of three samples of o x y h a e m o cyanin concentrated b y centrifugation and redispersed in saline containing 60 m M / l

3I

922

R . F . BURTON

of NaC1, 10 mM/l of CaC12 and 14 mM/l of MgC12. It represents the mean curve expressed in terms of a solution containing 17 g of haemocyanin/kg--the approximate concentration in blood containing 24 g of solutes/kg (cf. curve 1). It has been

3--

"r

g J 2o~

0

I

I

7

8

I

9

oH

FIG. 1. Buffer index curves: (1) Bicarbonate-free blood; 24 g solutes/kg. (2) Blood after removal of bicarbonate and haemocyanin; 6.5-7.1 g solutes/kg. (3)Haemocyanin in saline containing physiological concentrations of sodium, calcium and magnesium; 17 g haemocyanin/kg. corrected for the small amount of buffering not due to haemocyanin. Similar curves were obtained in each case, with a maximum buffer index at pH 7.5 of 0.18 _+ S.D. 0.02 m-equiv./pH per g. The titration curve of such haemocyanin from pH 5.2, the approximate isoelectric point (Redfield, 1934), is shown in Fig. 2. It is based on two samples and is expressed in terms of 1 g of haemocyanin. Three samples of blood were centrifuged at 8 x 107 cm/sec ~ (average) to yield for each a supernatant free of haemocyanin and another fraction rich in it. The pH of each was measured at 20°C after equilibration with carbon dioxide (when the haemocyanin would have been deoxygenated) and with mixtures of carbon dioxide and oxygen. The supernatant in each case behaved much like a simple solution of bicarbonate in that the concentration of bicarbonate plus carbonate did not change significantly with carbon dioxide tension. It averaged 27.6 mM/l. An average of 27 raM/1 was obtained by titration with hydrochloric acid, but the end-points were not as sharp as those obtained with simple bicarbonate solutions, presumably because small amounts of other buffers were present. In

923

BUFFERS I N T H E B L O O D OF T H E S N A I L

the presence of haemocyanin, the concentrations of bicarbonate increased with carbon dioxide tension and hydrogen ion activity. Changes in the ionization of haemoeyanin have been calculated from the changes in the amounts of bicarbonate 0.0

m

.c

O O'l--

-c

i

E 0.2--

E

g Q.

0"3--

I

I

6

7

I

8

pH

Fie. 2. Titration curve of oxyhaemocyanin in saline containing physiological concentrations of sodium, calcium and magnesium. Continuous line: titration with NaOH. Points: titrations of three samples with carbon dioxide. and carbonate present, assuming an ionic strength of 0.12, and are shown in Fig. 2. The vertical placing of each set of points is chosen to bring them near the curve, also shown, for the titration with sodium hydroxide of haemocyanin in saline.

Influence of salts on buffer index curves of oxyhaemocyanin Three samples of haemocyanin were concentrated by centrifugation, dialysed against distilled water for 5-9 days and then diluted with distilled water. Maximum buffering, of 0.13-0.16 m-equiv./pH per g, was found to occur at pH 8"5-8.7. Progressive addition of 3 M NaC1 to such solutions adjusted initially to pH 8"6 caused the pH to fall, the change in pH for a given ionic strength,/z, being equal to about ~tlogx0/z. Since the very low ionic strengths of the original solutions of haemocyanin could not be known exactly, the validity of this empirical relation at low concentrations is uncertain, but it applied from an ionic strength of 0.007 to one

924

R.F.

BURTON

of at least 0.2. After the addition of NaC1, to give concentrations of 0.06-0.22 M, peak buffering of NaOH moved to pH 7.8-7.9. Other samples of haemocyanin were concentrated and dialysed for 1-7 days against 0.04 or 0.1 M NaC1 and then diluted with the same. To aliquots of such solutions were added 1 M CaCI~, 1 M MgC12 or 3 M NaC1, raising the ionic strength to 0.1 or 0.16 and giving concentrations of alkaline earths of zero or 19.6 mM/1. The form and position of the buffer index curves were influenced by the presence or absence of calcium or magnesium, but for given ionic conditions similar curves were obtained with different samples of haemocyanin. The results obtained at the two ionic strengths were indistinguishable. The peak values for buffer index were as follows: in the absence of calcium and magnesium (six samples), 0-160 + S.D. 0.008 m-equiv./pH per g at pH 7.6-7.7; in the presence of 19.6 raM/1 of magnesium (three samples), 0.156 + S.D. 0-0045 m-equiv./pH per g at pH 7.5-7.6; in the presence of 19.6 mM/l of calcium (five samples), 0.171 + S.D. 0.0015 m-equiv./pH per g at pH 7.3-7.4. These results were obtained by titration with sodium hydroxide and, when calcium or magnesium was present in the solutions, similar curves were obtainable by titration with hydrochloric acid. This was true also of solutions lacking calcium and magnesium provided that the pH had not been above about 7"5. On the other hand, when titrations of the latter solutions were carried out with acid from a somewhat higher pH, for example pH 8 or above, maximum buffering occurred at a pH 0.2-0.3 units lower. On subsequent retitration with alkali, peak buffering occurred again at the higher value. Though this suggests a lack of equilibrium during the titrations, back-titrations followed a similar course whether performed immediately after titration with alkali or 20 min later. Moreover, the pH appeared stable following the addition of acid or alkali. In the same pH range as these anomalous results were obtained, there were changes in the blue colour of the haemocyanin. These involved a decrease in light extinction as the solutions became more alkaline, the decrease being inversely proportional to the fourth power of the wavelength over the range 400-700 nm. The changes could be reversed by the addition of calcium or magnesium. The addition of salts to solutions of haemocyanin in 0.1 M NaC1, adjusted initially to pH 7.5-7.6 under oxygen, caused them to become more acid (Fig. 3). Calcium had more effect than magnesium but both were much more effective than sodium. Calcium and magnesium were added as 1 M CaCI~ and 1 M MgC12 and sodium was added at the same ionic strength, i.e. 3 M, all solutions being at pH 7.6.

Haldane effects The results recorded so far were obtained with oxygenated haemocyanin. The effect of removing the oxygen from a solution of haemocyanin was to raise or lower the pH by a small amount that depended on the initial value (Fig. 4). The data shown here were obtained at 18-22°C with two samples of haemocyanin in saline, each pooled from five snails. From both of them were obtained two solutions differing in concentration of haemocyanin. Changes in pH were followed as the

BUFFERS

IN

THE

BLOOD

OF

THE

925

SNAIL

solutions were equilibrated alternately with nitrogen or with oxygen in nitrogen @ 0 2 = 37 m m Hg). As expected, the results did not indicate that the magnitude of the change in pH was influenced by the concentration of haemocyanin. Ionic strength 0.1 0"0 ~

0'2

015

I

NaCL

o-I -

0.41 0

[ 20 CO

or Mg,

J 40 mM/L.

FIG. 3. Changes in pH of two solutions of haemocyanin in 0.1 M NaCl o n addition of 1 M CaCI=, 1 M MgC12 and 3 M NaCI. Concentrations of sodium may be obtained from upper scale. 0 05~ • •

0

O

X

+

x+

T" 0.

c +

X + -005

I

o x

+

7.0

pH at

I 7.5

I

80

I

8.5

pO 2 of 3 7 m r n I-I(j

FIO. 4. Changes in pH of solutions of haemocyanin on deoxygenation in relation to the original pH at oxygen tension of 37 nun Hg. • and O : solutions, from one sample, containing 2"0% and 0"8% of haemocyanin respectively. + and x : solutions, from one sample, containing 2.0% and 1"3% of haemocyanin respectively.

Normal values of pH, pCO 2 and concentrations of bicarbonate The pH of blood from the haemocoel surrounding the digestive gland averaged 7.76 _+ S.D. 0.06 in twelve fed and active snails and 7-51 _+ S.D. 0"09 in ten aestivating specimens, the difference being highly significant ( P ~ 0.001). In eight

926

R . F . BURTON

active snails, the concentration of bicarbonate averaged 27 _+ S.D. 3 mM/l and the carbon dioxide tension 14 + S.D. 2 mm Hg, there being some correlation between the two ( P < 0.05). The equivalent figures for seven inactive snails were 21 _+ S.D. 4 raM/1 and 19 + S.D. 4 mm Hg. The differences in bicarbonate and pCO2 were both significant (P < 0.01) and would both contribute to the lower pH in aestivation. When blood was taken from both heart and haemocoel of each of six active snails, that from the heart was found to be more alkaline in every case, the difference averaging 0.12 + S.D. 0.03 pH units. DISCUSSION

Physicochemicalproperties of haemocyanin Anomalous results were obtained when oxyhaemocyanin was titrated in the presence of NaCI, but not of calcium or magnesium, in that the pH of maximum buffering varied according to whether acid or alkali was being added. This suggests a lack of equilibrium during the titrations, though this did not reveal itself through unstable pH readings. Furthermore, the buffer index curves obtained with NaOH were the same for different samples. The pH region in question, about 7.5-8.0, is that in which haemocyanin dissociates with increasing pH in the absence of calcium and magnesium and in which, therefore, light scattering decreases (Svedberg & Heyroth, 1929). The changes occurring in the appearance and light extinction of the solutions were such as would result from a decrease in light scattering and were reversible on addition of calcium. When the concentrations of all salts were reduced by dialysis of the haemocyanin against distilled water, maximum buffering of NaOH occurred at a higher pH, i.e. 8.5-8.7. Roche (1932) obtained results with purified haemocyanin similar with regard to the height and position of the peak of buffer index, but found a second peak at pH 7-0-7.1 that was not observed in the present study. Terroux (1936) found a peak at pH 8.2 with haemocyanin dialysed against distilled water. The influence of ionic strength on the pH of maximum buffering was illustrated also by the fall in pH as NaC1 was added to dialysed solutions of the protein. On the other hand, the buffer index curves at ionic strengths of 0.10 and 0.16, whether or not calcium or magnesium were present, were indistinguishable. At these ionic strengths, the pH of maximum buffering was 7.5-7.6 in the presence of 19.6 mM/l of magnesium and 7.3-7-4 in the presence of the same concentration of calcium. Addition of magnesium, and to a greater extent calcium, lowered the pH of solutions of haemocyanin more than did increasing the ionic strength the same amount with NaC1 (Fig. 3). It is likely that this was due to changes in the dissociation constants of buffer groups on the protein, but could also have involved an exchange of metal ions for protons. The samples of haemoeyanin used in this study were presumably mixtures of two forms in varying proportions (Brohult & Borgman, 1944) and yet buffer index curves obtained under given ionic conditions were consistent. Under approximately

BUFFERS I N T H E B L O O D O F T H E S N A I L

927

physiological conditions, haemocyanin was found to have a maximum buffer index of 0.17 m-equiv.]pH per g at a pH of 7.5, comparable to that of haemoglobin. If only one kind of ionizing group were involved, then there would have to be about fifteen of these per prosthetic group since each of the latter involves two copper atoms (Begemann, 1924) and the haemocyanin of H. poraatia contains about 0.254 per cent of copper (Burton, 1965a). As shown in Fig. 1, the buffer capacity of blood freed of bicarbonate approximates to that of haemocyanin, measured in the presence of sodium, calcium and magnesium in physiological concentrations, plus that due to other buffers. Terroux (1936) also found peak buffering in blood at pH 7"5, perhaps in the presence of some bicarbonate. Whether or not carbon dioxide has any direct effect on the ionization of haemocyanin, as by the formation of small amounts of carbamate, is not known, but titration with carbon dioxide yields results comparable to those obtained by titration with NaOH (Fig. 2). At pH values above 7.7, deoxygenation of solutions of haemocyanin was accompanied by a rise in p H - - a normal Haldane effect (Fig. 4). In more acid solutions the effect was reversed. In view of the small changes in pH involved, it is understandable that Wolvekamp & Kruyt (1944-47) did not detect any influence of oxygenation on their carbon dioxide curves. Haldane effects are necessarily associated with Bohr shifts and these, too, may be positive or negative. In fact the influence of pH on the oxygen dissociation curves of blood from H. poraatia is complex and depends on the oxygen tension, but, for the four samples studied at 15-20°C by Spoek et al. (1964), the oxygen tension at which half saturation occurred was highest at pH 7.6-7.9. Thus, reversal of the Bohr shift occurs at about the same pH as reversal of the Haldane effect. This is true also of horse haemoglobin (German & Wyman, 1937) though the reversal there is not in the physiological range of pH.

Physiological importance of blood buffers So far, the circumstances and range of variation in pH and bicarbonate concentration in the blood of H. pomatia have not been fully explored--nor the influence of the handling of the snails during sampling on carbon dioxide tensions and pH. However, in this limited study, samples of blood varied in pH from 7.4 to 8.0, a range consistent with earlier published figures quoted in the Introduction. Maximum buffering by haemoeyanin occurs, appropriately, within this range, at pH 7.5, the average pH of the blood in the aestivating specimens. In these, the low pH was in some cases apparently due both to an accumulation of carbon dioxide and to low levels of bicarbonate and, in others, due to just one of these. It is during aestivation, then, that haemocyanin will contribute most to the total buffer capacities of the blood, the pH being optimal and the contribution of bicarbonate minimal. Moreover, at this time, the concentration of haemocyanin is often raised through loss of water from the blood, even to 90 g/kg of water (Burton, 1965b). The total buffer capacity of the blood is the sum of the buffer capacities of haemoeyanin, flh, of bicarbonate,/~b (= 2.303 [HCOs']), and of the small amounts

928

R.F.

BURTON

of carbonate, fic (= 4.606 [COs"]) , and unidentified buffers, fix. In blood of pH 7.5, containing 20 mM/l of bicarbonate and 30 g[1 of haemocyanin, as might be found in inactive snails, the buffer capacity of the respiratory pigment as a proportion of the total, tih/(tih + fib + fic + fix), is 0.10. The buffering of changes in pCO2 is given by d(logt0pCOa) = d(pH)

1

~h+fic+fix= fib+tic

_ 1.12,

as compared with - 1.01 in the absence of haemoeyanin. Thus it is evident that haemocyanin does not generally contribute much to the stability of the pH of the blood. In any case, there may sometimes be as little as 7 g/kg water present in the blood of H. pomatia and the author has obtained blood quite free of it from Eobania

vermiculata. T A B L E 1 - - E F F E C T OF HAEMOCYANIN ON C O 2 TRANSPORT (NEGLECTING H A L D A N E EFFECTS) AND CONTRIBUTIONS OF DIFFERENT BUFFERS TO THE TOTAL BUFFER INDEX OF THE BLOOD IN ACTIVE AND AESTIVATING Helix pomatia

Active pH Bicarbonate, mM/l Haemocyanin, g/1

Aestivating

Representative concentrations 7-8 7"5 27 20 20 30

Increase in COa transport due to haemocyanin

x 2.1

x 2-7

Buffer indices as percentage of total Haemocyanin Bicarbonate Carbonate Other buffers

4"0 93"9 1"7 0"4

9"8 88.9 0'8 0"5

Concentrations of haemocyanin and bicarbonate are variable, but the calculations were based on typical values, as shown. Though haemocyanin contributes little to total buffer capacity, it does markedly facilitate the carriage of carbon dioxide (Table 1). Neglecting the Haldane effects, the amount of carbon dioxide released for a given fall in pCO2 can be calculated from the following equation. d(logl0pCOz)

= 2.30 [COd + Ha COd] +

fibCfih + Bx) + ½fi (fih + fix) + ½fibfi

fih+fi +fib+fi¢

where 4, = [CO~] + [H2COs] + [HCO~] + [CO~].

BUFFERS IN THE BLOOD OF THE SNAIL

929

I t is assumed here that no significant a m o u n t of carbamate is formed and that hydration of carbon dioxide reaches equilibrium despite the absence of carbonic anhydrase f r o m the blood (Wolverkamp & Kruyt, 1944-47). T h e magnitudes of the changes in tension and content of carbon dioxide as blood flows through the lung of an active snail are suggested b y the m e a n difference of 0.12 p H units between blood f r o m the heart and blood f r o m the haemocoel. F o r the typical active snail of T a b l e 1 the p C O 2 would fall f r o m 13.0 to 9.7 m m H g and the a m o u n t of carbon dioxide released would be 0.61 mM/1 (1.4 vol.%). REFERENCES

ASTRUP P. (1956) A simple electrometric technique for the determination of carbon dioxide tension in blood and plasma, total content of carbon dioxide in plasma, and bicarbonate content in "separated" plasma at a fixed carbon dioxide tension (40 mm Hg). ,Scand. jT. clin. Lab. Invest. 8, 33-43. BEGE~.~'qN H. (1924) Over de ademhalingsfianctie van haemocyanine. Thesis, Utrecht. Cited by REDFIELD A. C., COOLIDGET. & MONTGOMERYH. (1928) The respiratory proteins of the blood--II. The combining ratio of oxygen and copper in some bloods containing haemocyanin..7, biol. Chem. 76, 197-205. BROHULT S. & BORGMANK. (1944) L'h6mocyanine de Helix pomatia est-elle compos6e de deux esp6ces de molecules ? The ,Svedberg 1884-1944, p. 429. Almqvist & Wiksells, Uppsala. BURTON R. F. (1965a) Possible factors limiting the concentration of haemocyanin in the blood of the snail, Helix pomatia L. Can..~. Zool. 43, 433-438. BURTON R. F. (1965b) Sodium, potassium, and magnesium in the blood of the snail, Helix pomatia L. Physiol. Zo61. 38, 335-342. BURTON R. F. (1968) Ionic balance in the blood of Pulmonata. Comp. Bioehem. Physiol. 25, 509-516. DUVAL M. (1930) Concentration mol6culaire de sang de l'escargot, ses facteurs, ses variations. Influence de l'6tat d'activit6 de 1'animal. Annls Physiol. Physicochim. biol. 6, 346-364. GERMANB. & WYMANJ. (1937) The titration curves of oxygenated and reduced hemoglobin. ~. biol. Chem. 117, 533-550. HASTINGSA. B. & SENDROYJ. (1925) The effect of variation in ionic strength on the apparent first and second dissociation constants of carbonic acid..7, biol. Chem. 65, 445-455. KAUKOY. & AIROLAA. (1937) Die zweite Dissoziationskonstante der Kohlenslture. Z. phys. Chem. A 179, 307-313. NITZESCUI.-I. & COSMAI. (1927) La courbe de dissociation du CO s dans le liquide circulant d'Helix pomatia. C. r. Sdanc. Soc. Biol. 97, 1110-1111. RAFFY A. & FISCHER P.-H. (1935) Influence de la suroxyg6nation de l'atmosph6re sur le milieu int6rieur d'I-Ielix pomatia L. C. r. Sianc. Soc. Biol. 118, 15-17. REDFIELDA. C. (1934) The haemocyanins. Biol. ReD. 9, 175-212. ROCHE J. (1932) Recherches sur les propri6t6s physico-chimiques des h6mocyanines. Bull. Soe. Chim. biol. 14, 1032-1043. SIGGAARDANDERSEN0 . , ENGEL K., JORGRNSENK. & ASTRUPP. (1960) A micro method for determination of pH, carbon dioxide tension, base excess, and standard bicarbonate in capillary blood. Scand..~. clin. Lab. Invest. 12, 172-176. SOROKINA Z. A. (1965) Extra- and intra-cellular hydrogen ion activity determination in mollusc nerve cell ganglia. Zh. Evol. Biokhim. Fiz. 1, 343-350. SOROKL'qA Z. A. & ZELENSKAYAV. S. (1967) Peculiarities of electrolyte composition of molluscan haemolymph. Zh. Evol. Biokhim. Fiz. 3, 25-30.

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R.F.

BURTON

SPOEK G. L., BAKKEa H. & WOLVEKAMPH. P. (1964) Experiments on the haemocyaninoxygen equilibrium of the blood of the edible snail (Helix pomatia L.). Comp. Biochem. Physiol. 12, 209-221. SWDBm~O T. & I-Im~oTH F. F. (1929) The influence of the hydrogen-ion activity upon the stability of the hemocyanin of Helix pomatia, ft. Am. chem. Soc. 51, 550-561. TERROUX K. G. (1936) The buffering powers of natural and dialysed Helix pomatia serum. Biol. Bull., Woods Hole 70, 321-331. VORWOHL G. (1961) Zur Funktion der Excretionsorgane von Helix pomatia L. und Archachatina ventricosa Gould. Z. vergl. Physiol. 45, 12--49. WOLVam~P H. P. & K a u ~ W. (19A,a, A,7) Experiments on the carbon dioxide transport by the blood of the edible snail (Helix pomatia L.), the common crab (Cancer pagurus L.) and the common lobster (Homarus vulgaris M.E.). Archs nderl. Physiol. 28, 620-630.

Key Word Index--Helix pomatia; body fluids; haemocyanin; buffering; carbon dioxide transport.