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The oxygen affinity of carcinus mediterraneus haemocyanin-A calcium insensitive, dialysis induced decrease in O2 affinity
Comp. Biochem. Physiol., 1975, Vol. 52A, pp. 189 to 191. Pertlamon Press. Printed in Great Britain
THE OXYGEN AFFINITY OF C A R C I N U S M E D I T E R R A N E U S HAEMOCYANIN-A CALCIUM INSENSITIVE, DIALYSIS INDUCED DECREASE IN 02 AFFINITY R. R. HARRIS*, E. N. CHANTLERt, AND W. H. BANNISTER Department of Biology and Department of Physiology and Biochemistry, The Royal University of Malta, Msida, Malta (Received 9 July 1974)
Abstract--1. The Pso of whole untreated Carcinus mediterraneus haemolymph was found to be 9.5 -I- 0'8 (S.E.M.) mm Hg 02. 2. Dialysis against and dilution with Tris-HCI buffer, pH 7.6, resulted in a decreased 02 affinity. 3. This decrease in affinity was shown not to be due to low Ca 2÷ concentration or ionic strength. 4. Fractionation of whole haemolymph on Sephadex G-25 also resulted in a decrease in O2 affinity. 5. 02 affinity was found to be restored by the addition of haemolymph supernatant.
INTRODUCTION
MATERIALS AND METHODS
Carcinus mediterraneus (Cerniavsky) haemolymph was THE EFFECT of inorganic ions on the oxygen affinity of haemocyanins has been extensively studied collected from the base of a leg by hypodermic syringe. although in many cases it is difficult to differentiate After being allowed to clot at room temperature it was between the non-specific effect of ionic strength and filtered through glass wool and used immediately for oxygenation studies. These were carried out at 20°C using the specific requirement of a certain concentration methods described previously (Chantler et al., 1973). The of the divalent cations Ca 2+ and Mg 2÷. The latter absorbance of whole or dialysed haemolymph was meaare known to be important in co-operative interaction sured at 570 nm. Haemolymph to which sodium dithionite between 0 2 binding sites and the degree of aggrega- had been added was used here as a blank. For diluted tion of haemocyanin sub-units. haemolymph measurements were made at 340 nm, the releEarly work (Hogben, 1926; Hogben & Pinhey, vant buffer blanks being used. Dialysis of whole haemolymph was carried out against 1926, 1927) showed that the 02 affinity of Homarus haemocyanin was greatly increased by the presence 0'1 M Tris-HCl buffer, pH 7'6, containing 0'01 to 0"04M of high concentrations of Ca 2+ and Mg 2+. Low con- Ca 2+ at 6°C for 48 hr. Whole haemolymph was fractionated on a Sephadex centrations of these ions have been shown to cause G-25 column which had been equilibrated previously with a decreased affinity (Redmond, 1955; Larimer & Tris-HCl buffer, pH 7.6, containing 0.01 M Ca 2+. Samples Riggs, 1964; Er-E1 et al., 1972) whereas complete were collected on an LKB fraction collector, protein conremoval of the ions results in hyperbolic rather than centration of the eluate being monitored at 280 nm. Purifisigmoid 02 binding curves and greatly decreased 02 cation of haemocyanin was carried out as before (Chantler affinity (Chantler et al., 1973). Dialysis of haemo- et al., 1973). Haemolymph and buffer pH was determined before and cyanin solutions against Ca 2 + and Mg 2+ free hypotonic buffers caused a decrease in 02 affinity (Sted- after oxygenation using a Radiometer PHM 25 meter and man & Stedman, 1926; Hwang & Fung, 1970; Lar- G222C glass electrode. Haemolymph haemocyanin concentrations were estimated, using the specific extinction imer & Riggs, 1964). This effect could be partially coefficient at 280 nm for Carcinus haemocyanin determined reversed by the addition of Ca 2+ and Mg 2+ ions, in earlier work (Chantler et al., 1973) without correction and in some cases, 02 affinity completely restored for minor non-haemocyanin protein fractions in haemoby the addition of buffer salts. Djangmah & Grove lymph. (1971) have shown clearly that dilution of Crangon RESULTS blood with hypotonic buffers results in a lower (a) The effect o f dilution affinity and an increased buffer ionic strength caused The oxygen dissociation curves for freshly collected an increase in 02 affinity. We report here a dialysis whole haemolymph (mean haemocyanin coninduced oxygen affinity decrease in Carcinus mediterraneus haemocyanin which appears to be insensitive centration 36 mg/ml; mean pH 7.6 + 0.8 S.E.M.) to both Ca 2÷ ion concentration and ionic strength. were sigmoid with a mean P5o of 9"5 +__0'8 (S.E.M.) mm Hg 02. Dilution of whole haemolymph with 0.1 M Tris-* Present address: Department of Zoology, School of HCI buffer, pH7-6, containing 0.01 M Ca 2+ (total Biological Sciences, University of Leicester, Leicester LEI ionic strength 0'08; haemocyanin concentration 7RH, England. t Present address: Department of Human Reproduction 0.4 mg/ml) decreased the oxygen affinity. The Pso was and Obstetrics, University of Southampton, Southampton, 15-1 ___0.8 (S.E.M.) mm Hg 02. This decrease in 02 affinity was highly significant (P < 0.001) (Table 1). England. 189
190
R.R. HARRIS, E. N. CHANTLER AND W. H. BANNISTER
(b) The effect of dialysis Similarly, dialysis of whole haemolymph against the same pH 7"6 Tris-HCI buffer increased the PsQ to 16.1 + 2.1 (S.E.M.)mmHgOv This change was also significant (P < 0'01)(Table 1). Dialysis of whole haemolymph against buffer containing 0-02 and 0.04M Ca 2+ did not result in a higher 02 affinity.
(c) The effect of dialysis against high ionic strength
buffers The haemolymph Na + and C1 + concentrations in
C. mediterraneus are 489 and 540mM/1 respectively (Lucu et al., 1973). However dialysis of haemolymph against 0.1 M Tris-HCl buffer, pH 7-6, with 0-01 M Ca z+ and containing 0.4M NaC1 (ionic strength 0.480) resulted in a decrease in 02 affinity similar to that obtained in (b). The difference between the mean P5o values is not significant at the 5 ~ confidence level (0"05 < P < 0"10). The observed decreases in 02 affinity are not therefore due to changes in ionic strength (Table 1).
mocyanin was separated from whole haemolymph by ultracentrifugation and the supernatant (flee from haemocyanin) kept at 4°C. The haemocyanin pellet was washed, recentrifuged and resuspended in TrisHCI buffer, pH 7.6, containing 0"01 M Ca -'+ to give a protein concentration similar to that found in the original haemolymph (36 mg/ml). Oxygen dissociation curves determined at 570nm showed a reduced affinity with a Pso of 18.7 _+ 1.4 (S.E.M.)mm H g O v Subsequently the haemocyanin was recovered by recentrifugation, washed and resuspended in the original haemolymph supernatant. Haemocyanin concentrations and pH were adjusted to original haemolymph values. This reconstituted haemolymph showed an increased 02 affinity with a Pso of 8-0__+2'6 (S.E.M.)mmHg. Dilute reconstituted haemolymph (0-4 mg/ml protein) prepared similarly also showed a relatively high affinity with a Pso of 7.0 + 2'9 (S.E.M.) mm Hg. The Pso values of both reconstituted haemolymph solutions were not significantly different from that of freshly collected haemolymph (P > 0.10).
(d) Fractionation of whole haemolymph Gel permeation chromatography using Sephadex G-25 yielded a sharp 280 nm absorbance peak coincident with the void volume of the column. This peak was identified as haemocyanin. Its reduced affinity for 02, with a Pso of 18.9 + 2.5 (S.E.M.)mm Hg O2, was not the result of dilution with buffer during elution as shown by comparison with similarly diluted (1 in 4) fresh haemolymph. There was no significant differences at the 59/0 confidence level between Pso values of haemolymph treated in this way and those of (b) and (c). (P > 0"10).
(e) The reversal of the 02 affinity decrease The decrease in Oz affinity can be reversed by the addition of haemocyanin free haemolymph. Hae-
DISCUSSION
The Pso value determined for whole haemolymph of Carcinus mediterraneus is within the range previously reported for other marine decapod crustaceans (Redmond, 1971) and, in particular, is similar to the values found in C. maenas (Truchot, 1971; Buder & Taylor, 1973). It is apparent that measurements of 02 affinity of diluted or dialysed crustacean haemolymph may not necessarily apply to whole haemolymph. Redmond (1955) showed that these treatments resulted in displacement of the dissociation curves to the right. A higher Pso would thus be obtained. In C.
Table 1. Mean Ps0 (mm Hg 02) Whole haemolymph Haemolymph diluted with Tris-HCl buffer, pH 7.6 (with 0.01 M Ca 2+) Haemolymph dialysed against Tris-HCl buffer, pH 7.6 (with 0.01M Ca 2÷) Haemolymph dialysed as above, but buffer containing 0.02 M Ca -'+ Haemolymph dialysed as above, but buffer containing 0.04 M Ca 2÷ Haemolymph dialysed against Tris-HCl buffer, pH 7.6 with 0.01M Ca 2÷ and 0'4 M NaC1 Haemocyanin peak fraction from Sephadex G-25 gel chromatography Purified haemocyanin suspended in Tris-HCl buffer, pH 7.6 (with 0'01M Ca 2+) Reconstituted haemolymph (36 mg/ml protein) Reconstituted haemolymph (0'4 mg/ml protein) Haemolymph diluted 1 in 4 with Tris:HCl buffer, pH 7.6 (with 0.01 M Ca 2+)
S.E.M.
No. of determinations
9.5
0.8
10
15.1
0.8
8
16-1
2'1
8
17.1
1.9
5
16-8
0.9
5
20.1
1-5
6
18.9
2.5
4
18.7
1-4
5
8.0
1.6
5
7.0
1'9
5
9.7
0.8
4
Values of Pso for C. mediterraneus haemoeyanin in whole haemolymph and after various treatments.
The oxygen affinity of Carcinus mediterraneus mediterraneus dilution of haemolymph by a factor of about 100 results in a Pso 5-6 mm Hg above the whole haemolymph value. Dialysis results in a similar decrease in 02 affinity. It is apparent that a decrease in haemocyanin concentration per se with dilution is not the cause of this decreased affinity and this is supported by results using reconstituted haemolymph with low haemocyanin concentrations. In this respect our findings agree with those of Larimer & Riggs (1964) who showed that dissociation curves determined using the 340 and 570nm absorption peaks were identical in Procambarus similans. We were not able to carry out determinations at both wavelengths on the same sample, due to technical limitations. In C. mediterraneus we have found that both the dialysis and the dilution induced decrease in 0 2 affinity occurs in the presence of Ca 2+ ions, even at levels above the physiological mean (Chantler et al., 1973). We have shown previously that complete removal of Ca 2+ resulted in low O~ affinity and decreased co-operativity between 02 binding sites. Addition of Na ÷ and CI- up to physiological haemolymph concentrations does not reverse the affinity decrease. However, as shown by reconstitution of haemolymph from separated haemocyanin and haemolymph supernatant, a factor is present in the supernatant which restores 02 affinity, this apparently being neither haemolymph Ca 2+ nor overall ionic strength. The loss of this factor which occurs during gel fractionation on Sephadex G-25 or dialysis indicates a small molecule (mol. wt ~< 5000). This factor is not strongly associated with the haemocyanin molecule and is stable when separated, as shown by the results with reconstituted haemolymph. It does not affect the O2 binding equilibria which remain co-operative in its absence. Further comment on the identity of this factor awaits a more detailed investigation.
191
DJANGMAH J. S. & GROVE D. J. (1971) Oxygen affinity of haemocyanin in diluted blood of Crangon. Comp. Bigchem. Physiol. 38A, 461-464. ER-EL Z., SHAKLAIN. & DANIELE. (1972) Oxygen binding properties of haemocyanin from Levantina hierosolima. J. molec. Biol. 64, 341-352. HOGBEN L. T. (1926) Some observations on the dissociation of haemocyanin by the colorimetric method. Br. J. exp. Biol. 3, 225-238. HOGBEN L. T. & PINtmY K. F. (1926) A comparison between the dissociation of the haemocyanins of Helix and crustacea. Br. J. exp. Biol. 4, 203-214. HOGBEN L. T. & PINI-my K. F. (1927) Some observations on the haemocyanin of Limulus. Br. J. exp. Biol. 5, 55-65. HWANGJ. C. & FUNG C. P. (1970) Effect of calcium ions on oxygen equilibrium of haemocyanin of asiatic horseshoe crab Tachypleus tridentatus. Comp. Biochem. Physiol. 37, 573-579. LARIMER J. L. & RIGGS A. F. (1964) Properties of haemocyanins--l. The effect of calcium ions on the oxygen equilibrium of crayfish haemocyanin. Comp. Biochem. Physiol. 13, 35-46. Lucu C., SIEBERSD. & SPERLINGK. R. (1973) Comparison ofosmoregulation between Adriatic and North Sea Carcinus. Mar. Biol. 22, 85-95. REDMONDJ. R. (1955) The respiratory function of haemocyanin in crustacea, d. cell comp. Physiol. 46, 209-247. REDMOND J. R. (1971) Blood respiratory pigments--Arthropoda. In Chemical Zoology (Edited by FLORKIN M. & SCrlEER B. T.), Vol. 6 B, pp. 119-144. Academic Press, New York. STEDMANE. & STEDMANE, (1926) Haemocyanin--Part II. The influence of hydrogen ion concentration on the dissociation curve of oxyhaemocyanin from the blood of the common lobster Homarus oulgaris. Biochem. J. 20, 949-956. TAYLOR E. W. & BUTLERP. J. (1973) The behaviour and physiological responses of the shore crab Carcinus maenas during changes in environmental oxygen tension. Neth. J. Sea Res. 7, 496-505. TRUCHOT J. P. (1971) Fixation de roxygene par le serum de Carcinus maenas (L.) (crustace decapode brachyoure) C.r. hebd. Sdanc. Acad. Sci., Paris. 272, 984--987.
REFERENCES
CHANTLERE. N., HARRIS,R. R. & BANNISTERW. H. (1973) Oxygenation and aggregation properties of haemocyanin from Carcinus mediterraneus and Potamon edulis. Comp. Biochem. Physiol. 46A, 333-343.
Key Word Index--haemocyanin; Calcium; Carcinus mediterraneus.
THE OXYGEN AFFINITY OF C A R C I N U S M E D I T E R R A N E U S HAEMOCYANIN-A CALCIUM INSENSITIVE, DIALYSIS INDUCED DECREASE IN 02 AFFINITY R. R. HARRIS*, E. N. CHANTLERt, AND W. H. BANNISTER Department of Biology and Department of Physiology and Biochemistry, The Royal University of Malta, Msida, Malta (Received 9 July 1974)
Abstract--1. The Pso of whole untreated Carcinus mediterraneus haemolymph was found to be 9.5 -I- 0'8 (S.E.M.) mm Hg 02. 2. Dialysis against and dilution with Tris-HCI buffer, pH 7.6, resulted in a decreased 02 affinity. 3. This decrease in affinity was shown not to be due to low Ca 2÷ concentration or ionic strength. 4. Fractionation of whole haemolymph on Sephadex G-25 also resulted in a decrease in O2 affinity. 5. 02 affinity was found to be restored by the addition of haemolymph supernatant.
INTRODUCTION
MATERIALS AND METHODS
Carcinus mediterraneus (Cerniavsky) haemolymph was THE EFFECT of inorganic ions on the oxygen affinity of haemocyanins has been extensively studied collected from the base of a leg by hypodermic syringe. although in many cases it is difficult to differentiate After being allowed to clot at room temperature it was between the non-specific effect of ionic strength and filtered through glass wool and used immediately for oxygenation studies. These were carried out at 20°C using the specific requirement of a certain concentration methods described previously (Chantler et al., 1973). The of the divalent cations Ca 2+ and Mg 2÷. The latter absorbance of whole or dialysed haemolymph was meaare known to be important in co-operative interaction sured at 570 nm. Haemolymph to which sodium dithionite between 0 2 binding sites and the degree of aggrega- had been added was used here as a blank. For diluted tion of haemocyanin sub-units. haemolymph measurements were made at 340 nm, the releEarly work (Hogben, 1926; Hogben & Pinhey, vant buffer blanks being used. Dialysis of whole haemolymph was carried out against 1926, 1927) showed that the 02 affinity of Homarus haemocyanin was greatly increased by the presence 0'1 M Tris-HCl buffer, pH 7'6, containing 0'01 to 0"04M of high concentrations of Ca 2+ and Mg 2+. Low con- Ca 2+ at 6°C for 48 hr. Whole haemolymph was fractionated on a Sephadex centrations of these ions have been shown to cause G-25 column which had been equilibrated previously with a decreased affinity (Redmond, 1955; Larimer & Tris-HCl buffer, pH 7.6, containing 0.01 M Ca 2+. Samples Riggs, 1964; Er-E1 et al., 1972) whereas complete were collected on an LKB fraction collector, protein conremoval of the ions results in hyperbolic rather than centration of the eluate being monitored at 280 nm. Purifisigmoid 02 binding curves and greatly decreased 02 cation of haemocyanin was carried out as before (Chantler affinity (Chantler et al., 1973). Dialysis of haemo- et al., 1973). Haemolymph and buffer pH was determined before and cyanin solutions against Ca 2 + and Mg 2+ free hypotonic buffers caused a decrease in 02 affinity (Sted- after oxygenation using a Radiometer PHM 25 meter and man & Stedman, 1926; Hwang & Fung, 1970; Lar- G222C glass electrode. Haemolymph haemocyanin concentrations were estimated, using the specific extinction imer & Riggs, 1964). This effect could be partially coefficient at 280 nm for Carcinus haemocyanin determined reversed by the addition of Ca 2+ and Mg 2+ ions, in earlier work (Chantler et al., 1973) without correction and in some cases, 02 affinity completely restored for minor non-haemocyanin protein fractions in haemoby the addition of buffer salts. Djangmah & Grove lymph. (1971) have shown clearly that dilution of Crangon RESULTS blood with hypotonic buffers results in a lower (a) The effect o f dilution affinity and an increased buffer ionic strength caused The oxygen dissociation curves for freshly collected an increase in 02 affinity. We report here a dialysis whole haemolymph (mean haemocyanin coninduced oxygen affinity decrease in Carcinus mediterraneus haemocyanin which appears to be insensitive centration 36 mg/ml; mean pH 7.6 + 0.8 S.E.M.) to both Ca 2÷ ion concentration and ionic strength. were sigmoid with a mean P5o of 9"5 +__0'8 (S.E.M.) mm Hg 02. Dilution of whole haemolymph with 0.1 M Tris-* Present address: Department of Zoology, School of HCI buffer, pH7-6, containing 0.01 M Ca 2+ (total Biological Sciences, University of Leicester, Leicester LEI ionic strength 0'08; haemocyanin concentration 7RH, England. t Present address: Department of Human Reproduction 0.4 mg/ml) decreased the oxygen affinity. The Pso was and Obstetrics, University of Southampton, Southampton, 15-1 ___0.8 (S.E.M.) mm Hg 02. This decrease in 02 affinity was highly significant (P < 0.001) (Table 1). England. 189
190
R.R. HARRIS, E. N. CHANTLER AND W. H. BANNISTER
(b) The effect of dialysis Similarly, dialysis of whole haemolymph against the same pH 7"6 Tris-HCI buffer increased the PsQ to 16.1 + 2.1 (S.E.M.)mmHgOv This change was also significant (P < 0'01)(Table 1). Dialysis of whole haemolymph against buffer containing 0-02 and 0.04M Ca 2+ did not result in a higher 02 affinity.
(c) The effect of dialysis against high ionic strength
buffers The haemolymph Na + and C1 + concentrations in
C. mediterraneus are 489 and 540mM/1 respectively (Lucu et al., 1973). However dialysis of haemolymph against 0.1 M Tris-HCl buffer, pH 7-6, with 0-01 M Ca z+ and containing 0.4M NaC1 (ionic strength 0.480) resulted in a decrease in 02 affinity similar to that obtained in (b). The difference between the mean P5o values is not significant at the 5 ~ confidence level (0"05 < P < 0"10). The observed decreases in 02 affinity are not therefore due to changes in ionic strength (Table 1).
mocyanin was separated from whole haemolymph by ultracentrifugation and the supernatant (flee from haemocyanin) kept at 4°C. The haemocyanin pellet was washed, recentrifuged and resuspended in TrisHCI buffer, pH 7.6, containing 0"01 M Ca -'+ to give a protein concentration similar to that found in the original haemolymph (36 mg/ml). Oxygen dissociation curves determined at 570nm showed a reduced affinity with a Pso of 18.7 _+ 1.4 (S.E.M.)mm H g O v Subsequently the haemocyanin was recovered by recentrifugation, washed and resuspended in the original haemolymph supernatant. Haemocyanin concentrations and pH were adjusted to original haemolymph values. This reconstituted haemolymph showed an increased 02 affinity with a Pso of 8-0__+2'6 (S.E.M.)mmHg. Dilute reconstituted haemolymph (0-4 mg/ml protein) prepared similarly also showed a relatively high affinity with a Pso of 7.0 + 2'9 (S.E.M.) mm Hg. The Pso values of both reconstituted haemolymph solutions were not significantly different from that of freshly collected haemolymph (P > 0.10).
(d) Fractionation of whole haemolymph Gel permeation chromatography using Sephadex G-25 yielded a sharp 280 nm absorbance peak coincident with the void volume of the column. This peak was identified as haemocyanin. Its reduced affinity for 02, with a Pso of 18.9 + 2.5 (S.E.M.)mm Hg O2, was not the result of dilution with buffer during elution as shown by comparison with similarly diluted (1 in 4) fresh haemolymph. There was no significant differences at the 59/0 confidence level between Pso values of haemolymph treated in this way and those of (b) and (c). (P > 0"10).
(e) The reversal of the 02 affinity decrease The decrease in Oz affinity can be reversed by the addition of haemocyanin free haemolymph. Hae-
DISCUSSION
The Pso value determined for whole haemolymph of Carcinus mediterraneus is within the range previously reported for other marine decapod crustaceans (Redmond, 1971) and, in particular, is similar to the values found in C. maenas (Truchot, 1971; Buder & Taylor, 1973). It is apparent that measurements of 02 affinity of diluted or dialysed crustacean haemolymph may not necessarily apply to whole haemolymph. Redmond (1955) showed that these treatments resulted in displacement of the dissociation curves to the right. A higher Pso would thus be obtained. In C.
Table 1. Mean Ps0 (mm Hg 02) Whole haemolymph Haemolymph diluted with Tris-HCl buffer, pH 7.6 (with 0.01 M Ca 2+) Haemolymph dialysed against Tris-HCl buffer, pH 7.6 (with 0.01M Ca 2÷) Haemolymph dialysed as above, but buffer containing 0.02 M Ca -'+ Haemolymph dialysed as above, but buffer containing 0.04 M Ca 2÷ Haemolymph dialysed against Tris-HCl buffer, pH 7.6 with 0.01M Ca 2÷ and 0'4 M NaC1 Haemocyanin peak fraction from Sephadex G-25 gel chromatography Purified haemocyanin suspended in Tris-HCl buffer, pH 7.6 (with 0'01M Ca 2+) Reconstituted haemolymph (36 mg/ml protein) Reconstituted haemolymph (0'4 mg/ml protein) Haemolymph diluted 1 in 4 with Tris:HCl buffer, pH 7.6 (with 0.01 M Ca 2+)
S.E.M.
No. of determinations
9.5
0.8
10
15.1
0.8
8
16-1
2'1
8
17.1
1.9
5
16-8
0.9
5
20.1
1-5
6
18.9
2.5
4
18.7
1-4
5
8.0
1.6
5
7.0
1'9
5
9.7
0.8
4
Values of Pso for C. mediterraneus haemoeyanin in whole haemolymph and after various treatments.
The oxygen affinity of Carcinus mediterraneus mediterraneus dilution of haemolymph by a factor of about 100 results in a Pso 5-6 mm Hg above the whole haemolymph value. Dialysis results in a similar decrease in 02 affinity. It is apparent that a decrease in haemocyanin concentration per se with dilution is not the cause of this decreased affinity and this is supported by results using reconstituted haemolymph with low haemocyanin concentrations. In this respect our findings agree with those of Larimer & Riggs (1964) who showed that dissociation curves determined using the 340 and 570nm absorption peaks were identical in Procambarus similans. We were not able to carry out determinations at both wavelengths on the same sample, due to technical limitations. In C. mediterraneus we have found that both the dialysis and the dilution induced decrease in 0 2 affinity occurs in the presence of Ca 2+ ions, even at levels above the physiological mean (Chantler et al., 1973). We have shown previously that complete removal of Ca 2+ resulted in low O~ affinity and decreased co-operativity between 02 binding sites. Addition of Na ÷ and CI- up to physiological haemolymph concentrations does not reverse the affinity decrease. However, as shown by reconstitution of haemolymph from separated haemocyanin and haemolymph supernatant, a factor is present in the supernatant which restores 02 affinity, this apparently being neither haemolymph Ca 2+ nor overall ionic strength. The loss of this factor which occurs during gel fractionation on Sephadex G-25 or dialysis indicates a small molecule (mol. wt ~< 5000). This factor is not strongly associated with the haemocyanin molecule and is stable when separated, as shown by the results with reconstituted haemolymph. It does not affect the O2 binding equilibria which remain co-operative in its absence. Further comment on the identity of this factor awaits a more detailed investigation.
191
DJANGMAH J. S. & GROVE D. J. (1971) Oxygen affinity of haemocyanin in diluted blood of Crangon. Comp. Bigchem. Physiol. 38A, 461-464. ER-EL Z., SHAKLAIN. & DANIELE. (1972) Oxygen binding properties of haemocyanin from Levantina hierosolima. J. molec. Biol. 64, 341-352. HOGBEN L. T. (1926) Some observations on the dissociation of haemocyanin by the colorimetric method. Br. J. exp. Biol. 3, 225-238. HOGBEN L. T. & PINtmY K. F. (1926) A comparison between the dissociation of the haemocyanins of Helix and crustacea. Br. J. exp. Biol. 4, 203-214. HOGBEN L. T. & PINI-my K. F. (1927) Some observations on the haemocyanin of Limulus. Br. J. exp. Biol. 5, 55-65. HWANGJ. C. & FUNG C. P. (1970) Effect of calcium ions on oxygen equilibrium of haemocyanin of asiatic horseshoe crab Tachypleus tridentatus. Comp. Biochem. Physiol. 37, 573-579. LARIMER J. L. & RIGGS A. F. (1964) Properties of haemocyanins--l. The effect of calcium ions on the oxygen equilibrium of crayfish haemocyanin. Comp. Biochem. Physiol. 13, 35-46. Lucu C., SIEBERSD. & SPERLINGK. R. (1973) Comparison ofosmoregulation between Adriatic and North Sea Carcinus. Mar. Biol. 22, 85-95. REDMONDJ. R. (1955) The respiratory function of haemocyanin in crustacea, d. cell comp. Physiol. 46, 209-247. REDMOND J. R. (1971) Blood respiratory pigments--Arthropoda. In Chemical Zoology (Edited by FLORKIN M. & SCrlEER B. T.), Vol. 6 B, pp. 119-144. Academic Press, New York. STEDMANE. & STEDMANE, (1926) Haemocyanin--Part II. The influence of hydrogen ion concentration on the dissociation curve of oxyhaemocyanin from the blood of the common lobster Homarus oulgaris. Biochem. J. 20, 949-956. TAYLOR E. W. & BUTLERP. J. (1973) The behaviour and physiological responses of the shore crab Carcinus maenas during changes in environmental oxygen tension. Neth. J. Sea Res. 7, 496-505. TRUCHOT J. P. (1971) Fixation de roxygene par le serum de Carcinus maenas (L.) (crustace decapode brachyoure) C.r. hebd. Sdanc. Acad. Sci., Paris. 272, 984--987.
REFERENCES
CHANTLERE. N., HARRIS,R. R. & BANNISTERW. H. (1973) Oxygenation and aggregation properties of haemocyanin from Carcinus mediterraneus and Potamon edulis. Comp. Biochem. Physiol. 46A, 333-343.
Key Word Index--haemocyanin; Calcium; Carcinus mediterraneus.