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Automatic measurement of the oxygen affinity of Cancer magister hemocyanin
Comp. Biochem. Physiol., 1977. Vol. 57B, pp. 139 to 141. Pergamon Press, Printed in Great Britain
AUTOMATIC MEASUREMENT OF THE OXYGEN AFFINITY OF C A N C E R M A G I S T E R HEMOCYANIN H. W~OCMAN,* P. McMAttlLL AND H. S. MASON Department of Biochemistry, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A.
(Received 9 November 1976) Abstract--1. The P~o of Cancer magister hemocyanin in hemolymph was 40.0 mm at 25°C, nm,~ = 3.9; at I4°C, 15.0mm and 3.4; and at I0°C. 10.2mm and 3.3. 2. In the absence of Ca z + and Mg 2+, the Ps0 at 25°C was in the range of 150 to 250 mm, depending upon pH. 3, Our results show that temperature variation and pH variation may be important factors in the physiological regulation of oxygen delivery in this species. 4. The automatic recording device of Imai et al. (1970) previously used for the study of hemoglobins, was found to be eminently suitable for the study of 02 affinities of hemocyanins.
INTRODUCTION Hernocyanins are multimeric oxygen transporting proteins having binuclear Cu(I) clusters at the active sites of their protomers (Lontie & Vanquickenborn, 1974; Van Holde & Van Bruggen, 1971). They occur extensively among Arthropoda and Mollusca. Quantitative measurements of the oxygen affinity of hemocyanins extend back over at least 50 yr. Recent studies have shown that the degree of oxygenation of hemocyanins is affected by temperature, pH, the presence of inorganic ions, association~lissociation equilibria among protomers, and cooperative effects among the active sites (Lontie & Vanquickenborn, 1974; Van Holde & Van Bruggen, 1971). These studies were carried out with discontinuous spectrophotometric methods. The chemical properties of hemocyanin of the Pacific crab, Cancer magister, have been examined in some depth (Vanneste & Mason, 1966; Ellerton et al., 1970; Thomson et al., 1959; Carpenter & Van Holde, 1973, Moss et al., 1973; Loehr & Mason, 1971; Loehr & Mason, 1973; Schoot Uiterkamp and Mason, 1973; Schoot Uiterkamp et al., 1974), but its physiological properties are relatively undefined. In this study we examined the oxygen affinity of Cancer maffister hemocyanin under a variety of conditions of temperature, pH, and ionic medium, using for the first time--as far as we k n o w - t h e automatic recording technique described by Imai et al. (1970) for hemoglobin. We find that this method, applied to hemocyanin, gives very accurate results at both high and low POE values and allows a better determination of the Hill coefficient than previously possible.
crabs was collected on ice, diluted with an equal volume of cold 0.1 M phosphate buffer, pH. 7.0, and centrifuged for 20 min at 4°C, and 10,000 g to remove a small amount of sediment. The supernatant was centrifuged for 22 hr at 4°C, and 50,000 y. The hemocyanin pellet was dissolved in a small amount of buffer, and the preparation was sterilized by passage through a 0.22 micron Millipore filter. Hemolymph itself was prepared by centrifugation at 25,000 g for 20 rain at 4°C without dilution and sterilized as above.
Calcium, magnesium and copper analysis The three elements were determined using a Varian Techtron model AA-5 atomic absorption spectrophotometer with the appropriate hollow cathode lamps. Oxygen binding The oxygen equilibrium curves were recorded using the apparatus described in Imai et al. (1970). The percentage of oxygen saturation was determined on a Gilford model 2400 spectrophotometer by following the change of absorbance during deoxygenation at 345 nm for dilute hemocyanin solutions, and at 570nm for hemolymph. The oxygen partial pressure was continuously measured using a Clark oxygen electrode (Beckman 39065). The temperature of the sample was maintained within +0.1°C during measurements. Deoxygenation was performed by substituting free oxygen in the gas phase of the cell by nitrogen. When the hemocyanin was not fully saturated at 1.0atm of oxygen, the initial value was extrapolated to infinite 02 pressure by plotting 1/A vs 1/pO2. No denaturation occurred during the measurement, as indicated by an absence of time-dependent absorbancy increase when the sample was fully deoxygenated. Hill plots were computed from selected points on continuous experimental curves using a FOCAL-11 program written for a PDP-11 computer.
M~TERIALS AND M E T H O D S
Hemocyanin and hemolymph preparations The hemocyanin was prepared according to Thomson et al. (1959). The hemolymph of lively Cancer magister
RESULTS AND DISCUSSION
Oxygen binding in physiological conditions In vivo, Cancer ma#ister hemocyanin is mainly in the form of a 25 S dodecamer of the 5 S subunits, * Permanent address: Institut de Pathologie Molecu- together with a small amount of 16 S hexamers laire~Groupe U 15 INSERM-24 R. du Faubourg St. (Thomson et al., 1959). In order to measure the oxygen affinity under these conditions we studied an Jacques, 75014 Paris, France. 139 '
H. WAJCMAN,P. McMAHILL AND H. S. Mason
140
.@
O®
#,H/ g
.,./
7/ ,~*
-2
I
-~
l . . . .
I
I
0
i
2
loq pO 2 Fig. 1. Oxygen affinity of undiluted Cancer magister hemolymph as a function of temperature. Experimental conditions: pH 7.55, Ca 2+, 15.4 x 10 3M; Mg 2*, 18.0 x 10-3 M. (A) 10C, (BI 14'C, (C) 25C. undiluted hemolymph sample. Its characteristics were the following: Ca 2+ 15.4raM, Mg 2+ 18.0raM, pH 7.55 and Asvs = 0.374. The results obtained at different temperatures are reported in Fig. 1 and Table 1. From the comparison of these data with the oxygen affÉnity parameters of the crayfish (Procambarus simulans) hemocyanin (Larimer & Riggs, 1964), or of the shrimp (Callianassa caliJbrniensis) hemocyanin (Miller & Van Holde, 1974) measured in similar conditions, it appears that the P5o value is much higher in the case of Cancer magister. By raising the temperature from 15 to 2YC, the Pso of the shrimp or of the crayfish hemocyanin increased less than two fold and remained high. The change is about 50~o larger for Cancer magister. Temperature variations must therefore be, for this poikilothermic species, one of the factors involved in the physiological regulation of the oxygen delivery.
obtained near pH 7.0. Since at this pH, in the absence of divalent ions, the molecule is completely in its 25 S form (Thomson et at., 1959), the hypothesis that there is a parallelism between the decrease of the oxygen affinity and the state of aggregation is supported. Our data demonstrate also. as it was pointed out in the study of Palinurus interruplus hemocyanin (Kuiper el al., 1975) that the binding of oxygen is cooperative even in the absence of Ca 2. and Mg a , The n values were between 3 and 4. indicating a large homotropic effect. In the presence of Ca e~ and Mg-" the phenomenon observed was similar to that described by Larimer & Riggs 11964) for crayfish hemocyanin. The slope of the pH dependence curve is much steeper at alkaline pH. Of particular physiological interest is the magnitude of this slope and its maximum value, close to 1.0 between pH 7.0 and 8.0. These results indicate that in the presence of the divalent ions, a slight modification of pH leads to a large change of Pso. Since similar studies on other arthropodal hemocyanins (Lontie & Vanquickenborn. 1974: Van Holde & Van Bruggen, 1971) were conducted under different experimental conditions, it is not possible to compare our results exactly to those of the literature: nevertheless, for all pH values, the Ps~ of Cancer maqister appears high. The effect of Ca-'+ and Mg e' was studied at pH 7.3 on a hemocyanin sample previously dialyzed against Tris HCI buffer. The results are shown in Fig. 3. At this pH, the extent of the change produced by Mg2" or Ca-' + is moderate, By increasing the Mg :~ concentration from 0 to 50 mM there is a 33'!,, decrease of Pso. C a-'+ is more effective than Mg: . The same modification of concentration leads to a 60",, decrease of Pso. With Mg 2+. tit low concentration, a slight but significant increase of the Ps0 is first observed, and the value of the Pso decreases only after further increase of the Mg e ' concentration. A possible explanation of this fact may be that the divalent loll first shifts the equilibrium towards the 25 S form and only after this step acts as an heterotropic ligand shifting the dodecamer molecule from a low to a high oxygen affinity conformation.
Effect of pH and inorganic ions Using a purified hemocyanin (total copper--4.08 raM, EPR detectable copper less than 1°o, Aa,,0/Azs o = 0.218), we studied the effect of pH and of Ca 2+ and Mg 2+ concentrations at 25°C independently. The action of pH was measured in phosphate buffer, in the absence of Ca z + and Mg 2+, on a dialyzed sample of hemocyanin. Under these conditions, as shown in Fig. 2, the Pso is very high (in the range of 150 to 250mmHg), and a maximum value is Table 1. Oxygen affinity of Cancer maoister hemocyanin as a function of temperature
P50
.j.f'
mm Hg
n,,,~
10~'C 14~C 25C
10.2 15.0 4O.0
3.3 3.4 3.9
"X~.
\
q~2.0
• ~x X. ~o \
•~.
\ "\. \
\ \X
'\0,
\
\
1,0
'-o 6.5
Temperature
XN.
xj "
7.0
75
8.0
85
pH
Fig. 2. Log Ps0 dependence on ptt of ('am'er ma~jister hemocyanin. Crosses: phosphate 0.1 M buffers, 25 ( , without divalent ions. Circles: Tris-HC1 0.1 M, Ca 2 10× 10 3 M : M g 2" 50 × 10 -~M. 25 ('.
Oxygen affinity of Cancer magister hemocyanin
r
2.s t-/ I~'0
"N.X
X
"'-x....
~
f
M.++
""
l
' ' ' - - . - - - - . ca++
2.0I
"~'~o
"~ 0..,...
t
i
1
l
~
l
I0
20
30
40
50
[IO"M]
Ca*÷orMg÷+
Fig. 3. Effects of [Ca z÷] and [-Mg2+] on Ps0 of Cancer magister hemocyanin. Tris-HC1, 0.1 M, pH 7.3, 25°C. The results of this study suggest, in vivo, the oxygen delivery function of Cancer magister hemocyanin is regulated mainly by physico-chemical factors. Apparently there is no need for a small organic anion like organic phosphates which modify hemoglobins (but an organic cation could compete with Ca 2÷ or Mg2÷). The automatic recording device of Imai et' al. (1970) is eminently suitable for the study of O2 affinities of hemocyanins.
Acknowledgements--We wish to thank Dr. R. T. Jones for the use of his automatic recording oxygen affinity measuring instrument. H. W. thanks NATO for a long-term fellowship. This study was supported in part by a grant, NIH, AM 17850, to Dr. R. T. Jones, and by grants AM 0718 from the National Institutes of Arthritis and Metabolic Diseases, and BC-1M, from the American Cancer Society.
REFERENCES
CARPENTERD. E. & VAN HOLDEK. E. (1973) Amino acid composition, amino-terminal analysis, and subunit structure of Cancer magister hemocyanin. Biochemistry 12, 2231-2238. ELLERTON H. D., CARPENTERD. E. & VAN HOLDE K. E. (1970) Physical studies of hemocyanins--V. Characteri-
141
zation and sub-unit structure of the hemocyanin Cancer magister. Biochemistry 9, 2225-2232. IMAI K., MORIMOTOH., KOTANI M., WATARI H., HIRATA W. & KUROOA M. (1970) Studies on the function of abnormal hemoglobins--I. An improved method for automatic measurement of the oxygen equilibrium curve of hemoglobin. Biochim. biophys. Acta 200. 189 196. KUIPER H. A., GAASTRA W., BEINTEMA J, J., VAN BRUGGEN E. F. J., SCHEPMANA. M. H. & DRENTHJ. (1975) Subunit composition, X-ray diffraction, amino acid analysis and oxygen binding behaviour of Panulirus interreptus hemocyanin. J. molec. Biol. 99, 619~529. LARIMERJ. L. & RIGGS A. F. (1964) Properties of hemocyanins--I. The effect of calcium ions on the oxygen equilibrium of crayfish hemocyanin. Cutup. Biochem. Physiol. 13, 35-46. LOEHRJ. S. & MASONH. S. (1971) Dimorphism of Cancer magister hemocyanin subunits. EMBO Conference: Hemocyanins, Louvain, Belgium. LoErm J. S. & MASONH. S. (1973) Dimorphism of Cancer magister hemocyanin subunits. Biochem. biophys. Res. Commun. 51, 741-745. LONTIE R. & VANQUICKENBORN L. (1974) Metal Ions in Biological Systems, p. 183. Marcel Dekker, New York. MILLER K. & VAN HOLDE K. E. Oxygen binding by Callianassa californiensis hemocyanin. Biochemistry 13, 1668-1674. MOSS T. L., GOULD D. C., EHRENBERGA., LOEHR J. S. & MASON H. S. (1973) Magnetic properties of Cancer magister hemocyanin. Biochemistry 12. 2444-2449. SCHOOTUITERKA~d'A. J. M. & MASON H. S. (1973) Magnetic dipole-dipole coupled Cu(II) pairs in nitric oxidetreated tyrosinase: A structural relationship between the active sites of tyrosinase and hemocyanin. Proc. natn. Acad. Sci. U.S.A. 70, 993-996. SCHOOT UITERKAMP A, J. M., VAN DER DEEN H., BERENDSEN H. C. J. & BOAS J. F. (1974) Computer simulation of the EPR spectra of mononuclear and dipolar coupled Cu(II) ions in nitric oxide- and nitrite-treated hemocyanins and tyrosinase. Biochim. hiophys. Acta 372. 407-425. Tr~OMSONL. C. G., HINES M. & MASON H. S. (1959) On the binding of copper by hemocyanin. Archs Biochem. Biophys. 83, 88-95. VAN HOLDE K. E. VAN BRUGGEN E. F. J. (1971) Subunits in Biological Systems, p. 1. Marcel Dekker, New York. VANNESTEW. & MASONH. S. (1966) The Biochemistry of Copper, p. 465. Academic Press, New York.
AUTOMATIC MEASUREMENT OF THE OXYGEN AFFINITY OF C A N C E R M A G I S T E R HEMOCYANIN H. W~OCMAN,* P. McMAttlLL AND H. S. MASON Department of Biochemistry, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201, U.S.A.
(Received 9 November 1976) Abstract--1. The P~o of Cancer magister hemocyanin in hemolymph was 40.0 mm at 25°C, nm,~ = 3.9; at I4°C, 15.0mm and 3.4; and at I0°C. 10.2mm and 3.3. 2. In the absence of Ca z + and Mg 2+, the Ps0 at 25°C was in the range of 150 to 250 mm, depending upon pH. 3, Our results show that temperature variation and pH variation may be important factors in the physiological regulation of oxygen delivery in this species. 4. The automatic recording device of Imai et al. (1970) previously used for the study of hemoglobins, was found to be eminently suitable for the study of 02 affinities of hemocyanins.
INTRODUCTION Hernocyanins are multimeric oxygen transporting proteins having binuclear Cu(I) clusters at the active sites of their protomers (Lontie & Vanquickenborn, 1974; Van Holde & Van Bruggen, 1971). They occur extensively among Arthropoda and Mollusca. Quantitative measurements of the oxygen affinity of hemocyanins extend back over at least 50 yr. Recent studies have shown that the degree of oxygenation of hemocyanins is affected by temperature, pH, the presence of inorganic ions, association~lissociation equilibria among protomers, and cooperative effects among the active sites (Lontie & Vanquickenborn, 1974; Van Holde & Van Bruggen, 1971). These studies were carried out with discontinuous spectrophotometric methods. The chemical properties of hemocyanin of the Pacific crab, Cancer magister, have been examined in some depth (Vanneste & Mason, 1966; Ellerton et al., 1970; Thomson et al., 1959; Carpenter & Van Holde, 1973, Moss et al., 1973; Loehr & Mason, 1971; Loehr & Mason, 1973; Schoot Uiterkamp and Mason, 1973; Schoot Uiterkamp et al., 1974), but its physiological properties are relatively undefined. In this study we examined the oxygen affinity of Cancer maffister hemocyanin under a variety of conditions of temperature, pH, and ionic medium, using for the first time--as far as we k n o w - t h e automatic recording technique described by Imai et al. (1970) for hemoglobin. We find that this method, applied to hemocyanin, gives very accurate results at both high and low POE values and allows a better determination of the Hill coefficient than previously possible.
crabs was collected on ice, diluted with an equal volume of cold 0.1 M phosphate buffer, pH. 7.0, and centrifuged for 20 min at 4°C, and 10,000 g to remove a small amount of sediment. The supernatant was centrifuged for 22 hr at 4°C, and 50,000 y. The hemocyanin pellet was dissolved in a small amount of buffer, and the preparation was sterilized by passage through a 0.22 micron Millipore filter. Hemolymph itself was prepared by centrifugation at 25,000 g for 20 rain at 4°C without dilution and sterilized as above.
Calcium, magnesium and copper analysis The three elements were determined using a Varian Techtron model AA-5 atomic absorption spectrophotometer with the appropriate hollow cathode lamps. Oxygen binding The oxygen equilibrium curves were recorded using the apparatus described in Imai et al. (1970). The percentage of oxygen saturation was determined on a Gilford model 2400 spectrophotometer by following the change of absorbance during deoxygenation at 345 nm for dilute hemocyanin solutions, and at 570nm for hemolymph. The oxygen partial pressure was continuously measured using a Clark oxygen electrode (Beckman 39065). The temperature of the sample was maintained within +0.1°C during measurements. Deoxygenation was performed by substituting free oxygen in the gas phase of the cell by nitrogen. When the hemocyanin was not fully saturated at 1.0atm of oxygen, the initial value was extrapolated to infinite 02 pressure by plotting 1/A vs 1/pO2. No denaturation occurred during the measurement, as indicated by an absence of time-dependent absorbancy increase when the sample was fully deoxygenated. Hill plots were computed from selected points on continuous experimental curves using a FOCAL-11 program written for a PDP-11 computer.
M~TERIALS AND M E T H O D S
Hemocyanin and hemolymph preparations The hemocyanin was prepared according to Thomson et al. (1959). The hemolymph of lively Cancer magister
RESULTS AND DISCUSSION
Oxygen binding in physiological conditions In vivo, Cancer ma#ister hemocyanin is mainly in the form of a 25 S dodecamer of the 5 S subunits, * Permanent address: Institut de Pathologie Molecu- together with a small amount of 16 S hexamers laire~Groupe U 15 INSERM-24 R. du Faubourg St. (Thomson et al., 1959). In order to measure the oxygen affinity under these conditions we studied an Jacques, 75014 Paris, France. 139 '
H. WAJCMAN,P. McMAHILL AND H. S. Mason
140
.@
O®
#,H/ g
.,./
7/ ,~*
-2
I
-~
l . . . .
I
I
0
i
2
loq pO 2 Fig. 1. Oxygen affinity of undiluted Cancer magister hemolymph as a function of temperature. Experimental conditions: pH 7.55, Ca 2+, 15.4 x 10 3M; Mg 2*, 18.0 x 10-3 M. (A) 10C, (BI 14'C, (C) 25C. undiluted hemolymph sample. Its characteristics were the following: Ca 2+ 15.4raM, Mg 2+ 18.0raM, pH 7.55 and Asvs = 0.374. The results obtained at different temperatures are reported in Fig. 1 and Table 1. From the comparison of these data with the oxygen affÉnity parameters of the crayfish (Procambarus simulans) hemocyanin (Larimer & Riggs, 1964), or of the shrimp (Callianassa caliJbrniensis) hemocyanin (Miller & Van Holde, 1974) measured in similar conditions, it appears that the P5o value is much higher in the case of Cancer magister. By raising the temperature from 15 to 2YC, the Pso of the shrimp or of the crayfish hemocyanin increased less than two fold and remained high. The change is about 50~o larger for Cancer magister. Temperature variations must therefore be, for this poikilothermic species, one of the factors involved in the physiological regulation of the oxygen delivery.
obtained near pH 7.0. Since at this pH, in the absence of divalent ions, the molecule is completely in its 25 S form (Thomson et at., 1959), the hypothesis that there is a parallelism between the decrease of the oxygen affinity and the state of aggregation is supported. Our data demonstrate also. as it was pointed out in the study of Palinurus interruplus hemocyanin (Kuiper el al., 1975) that the binding of oxygen is cooperative even in the absence of Ca 2. and Mg a , The n values were between 3 and 4. indicating a large homotropic effect. In the presence of Ca e~ and Mg-" the phenomenon observed was similar to that described by Larimer & Riggs 11964) for crayfish hemocyanin. The slope of the pH dependence curve is much steeper at alkaline pH. Of particular physiological interest is the magnitude of this slope and its maximum value, close to 1.0 between pH 7.0 and 8.0. These results indicate that in the presence of the divalent ions, a slight modification of pH leads to a large change of Pso. Since similar studies on other arthropodal hemocyanins (Lontie & Vanquickenborn. 1974: Van Holde & Van Bruggen, 1971) were conducted under different experimental conditions, it is not possible to compare our results exactly to those of the literature: nevertheless, for all pH values, the Ps~ of Cancer maqister appears high. The effect of Ca-'+ and Mg e' was studied at pH 7.3 on a hemocyanin sample previously dialyzed against Tris HCI buffer. The results are shown in Fig. 3. At this pH, the extent of the change produced by Mg2" or Ca-' + is moderate, By increasing the Mg :~ concentration from 0 to 50 mM there is a 33'!,, decrease of Pso. C a-'+ is more effective than Mg: . The same modification of concentration leads to a 60",, decrease of Pso. With Mg 2+. tit low concentration, a slight but significant increase of the Ps0 is first observed, and the value of the Pso decreases only after further increase of the Mg e ' concentration. A possible explanation of this fact may be that the divalent loll first shifts the equilibrium towards the 25 S form and only after this step acts as an heterotropic ligand shifting the dodecamer molecule from a low to a high oxygen affinity conformation.
Effect of pH and inorganic ions Using a purified hemocyanin (total copper--4.08 raM, EPR detectable copper less than 1°o, Aa,,0/Azs o = 0.218), we studied the effect of pH and of Ca 2+ and Mg 2+ concentrations at 25°C independently. The action of pH was measured in phosphate buffer, in the absence of Ca z + and Mg 2+, on a dialyzed sample of hemocyanin. Under these conditions, as shown in Fig. 2, the Pso is very high (in the range of 150 to 250mmHg), and a maximum value is Table 1. Oxygen affinity of Cancer maoister hemocyanin as a function of temperature
P50
.j.f'
mm Hg
n,,,~
10~'C 14~C 25C
10.2 15.0 4O.0
3.3 3.4 3.9
"X~.
\
q~2.0
• ~x X. ~o \
•~.
\ "\. \
\ \X
'\0,
\
\
1,0
'-o 6.5
Temperature
XN.
xj "
7.0
75
8.0
85
pH
Fig. 2. Log Ps0 dependence on ptt of ('am'er ma~jister hemocyanin. Crosses: phosphate 0.1 M buffers, 25 ( , without divalent ions. Circles: Tris-HC1 0.1 M, Ca 2 10× 10 3 M : M g 2" 50 × 10 -~M. 25 ('.
Oxygen affinity of Cancer magister hemocyanin
r
2.s t-/ I~'0
"N.X
X
"'-x....
~
f
M.++
""
l
' ' ' - - . - - - - . ca++
2.0I
"~'~o
"~ 0..,...
t
i
1
l
~
l
I0
20
30
40
50
[IO"M]
Ca*÷orMg÷+
Fig. 3. Effects of [Ca z÷] and [-Mg2+] on Ps0 of Cancer magister hemocyanin. Tris-HC1, 0.1 M, pH 7.3, 25°C. The results of this study suggest, in vivo, the oxygen delivery function of Cancer magister hemocyanin is regulated mainly by physico-chemical factors. Apparently there is no need for a small organic anion like organic phosphates which modify hemoglobins (but an organic cation could compete with Ca 2÷ or Mg2÷). The automatic recording device of Imai et' al. (1970) is eminently suitable for the study of O2 affinities of hemocyanins.
Acknowledgements--We wish to thank Dr. R. T. Jones for the use of his automatic recording oxygen affinity measuring instrument. H. W. thanks NATO for a long-term fellowship. This study was supported in part by a grant, NIH, AM 17850, to Dr. R. T. Jones, and by grants AM 0718 from the National Institutes of Arthritis and Metabolic Diseases, and BC-1M, from the American Cancer Society.
REFERENCES
CARPENTERD. E. & VAN HOLDEK. E. (1973) Amino acid composition, amino-terminal analysis, and subunit structure of Cancer magister hemocyanin. Biochemistry 12, 2231-2238. ELLERTON H. D., CARPENTERD. E. & VAN HOLDE K. E. (1970) Physical studies of hemocyanins--V. Characteri-
141
zation and sub-unit structure of the hemocyanin Cancer magister. Biochemistry 9, 2225-2232. IMAI K., MORIMOTOH., KOTANI M., WATARI H., HIRATA W. & KUROOA M. (1970) Studies on the function of abnormal hemoglobins--I. An improved method for automatic measurement of the oxygen equilibrium curve of hemoglobin. Biochim. biophys. Acta 200. 189 196. KUIPER H. A., GAASTRA W., BEINTEMA J, J., VAN BRUGGEN E. F. J., SCHEPMANA. M. H. & DRENTHJ. (1975) Subunit composition, X-ray diffraction, amino acid analysis and oxygen binding behaviour of Panulirus interreptus hemocyanin. J. molec. Biol. 99, 619~529. LARIMERJ. L. & RIGGS A. F. (1964) Properties of hemocyanins--I. The effect of calcium ions on the oxygen equilibrium of crayfish hemocyanin. Cutup. Biochem. Physiol. 13, 35-46. LOEHRJ. S. & MASONH. S. (1971) Dimorphism of Cancer magister hemocyanin subunits. EMBO Conference: Hemocyanins, Louvain, Belgium. LoErm J. S. & MASONH. S. (1973) Dimorphism of Cancer magister hemocyanin subunits. Biochem. biophys. Res. Commun. 51, 741-745. LONTIE R. & VANQUICKENBORN L. (1974) Metal Ions in Biological Systems, p. 183. Marcel Dekker, New York. MILLER K. & VAN HOLDE K. E. Oxygen binding by Callianassa californiensis hemocyanin. Biochemistry 13, 1668-1674. MOSS T. L., GOULD D. C., EHRENBERGA., LOEHR J. S. & MASON H. S. (1973) Magnetic properties of Cancer magister hemocyanin. Biochemistry 12. 2444-2449. SCHOOTUITERKA~d'A. J. M. & MASON H. S. (1973) Magnetic dipole-dipole coupled Cu(II) pairs in nitric oxidetreated tyrosinase: A structural relationship between the active sites of tyrosinase and hemocyanin. Proc. natn. Acad. Sci. U.S.A. 70, 993-996. SCHOOT UITERKAMP A, J. M., VAN DER DEEN H., BERENDSEN H. C. J. & BOAS J. F. (1974) Computer simulation of the EPR spectra of mononuclear and dipolar coupled Cu(II) ions in nitric oxide- and nitrite-treated hemocyanins and tyrosinase. Biochim. hiophys. Acta 372. 407-425. Tr~OMSONL. C. G., HINES M. & MASON H. S. (1959) On the binding of copper by hemocyanin. Archs Biochem. Biophys. 83, 88-95. VAN HOLDE K. E. VAN BRUGGEN E. F. J. (1971) Subunits in Biological Systems, p. 1. Marcel Dekker, New York. VANNESTEW. & MASONH. S. (1966) The Biochemistry of Copper, p. 465. Academic Press, New York.