- Email: [email protected]
An investigation of the ionic calcium assay based on the jellyfish protein aequorin
ANALYTICAL
An
BIOCHEMISTRY
investigation on the
58,
479484
(1974)
of the
Ionic
Jellyfish
Protein
Calcium
for
,J. I. NICHOLLS, T. YODA
Research in Oral Biology and Anesthesiology Research University of Washington, Seattle, Washington Received
July
9, 1973;
accepted
Based
Aequorin
I<. T. IZUTSU, S. P. FELTON, I. A. SIEGEL, J. CRAWFORD, ,J. McGOUGH, AND W. Center
Assay
October
Center,
3, 1973
The ionic calcium assay based on the luminescence of the jellyfish protein aequorin was studied. A rapid mixing device was found to be necessary for good reproducibility. The accuracy of the procedure described could depend on the association constants of the calcium chelating agents involved in the test solutions. INTRODUCTION
Shimomura et al. (1) have described a method that may be capable of measuring the concentration of ionic calcium in serum. This procedure is based on the luminescence of the jellyfish protein aequorin. Our previous attempts (2) to utilize this procedure were only partially successful because of the difficulty in achieving rapid, consistent mixing and because the serum samples were diluted to slow the reaction to a measurable rate. We report here on our further studies of this assay. In particular, we have found that a rapid mixing device is necessary for consistent results and that the accuracy of the assay depends on the association constant of the calcium buffer used in the test solution. METHODS
The aequorin was prepared as descri,bed elsewhere (2,3) except that the sephadex column chromatography was done at pH 7.5. The specific activity of the aequorin used in these studies was approximately 1.5 X 1Ol5 photons/mg protein. This is a third of the value reported by Shimomura and Johnson (4) for pure aequorin and about eight times higher than the value reported by Baker et al. (5) for the aequorin used in their physiological studies. This specific activity is also approximately the same as the value reported by Shimomura et al. (1) for the aequorin used in their serum studies and also within the range of specific activities given by Johnson and Shimomura (6) as being adequate for most purposes. Further chromatography (on DEAE) resulted in a higher Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
479 Inc. reserved.
480
IZUTSU
ET
.4L.
specific activity, but this was offset by a decreased yield which made the procedure impractical when large amounts of aequorin are necessary as has been previously recognized (5,6). The major contaminant of our aequorin was probably the green protein (7) since there was a detectable 510 nm fluorescence with 460 excitation. The primary calibration solutions contained between 0 and 3 mM CaCl, as well as 150 mM NaCl. The addition of 10 mM Tris buffer (pH 7.4) to these solutions had no effect on the calibration curves. The serum calibration standards contained 0.75 mM MgCl*, 10 mM Tris buffer (pH 7.4)) and 5 mM KC1 as well as NaCl and CaCl, since magnesium is known to affect the rate of the aequorin-calcium reaction (7). Saliva was collected under stimulated conditions (while sucking sour lemon drops) by the method of Carlson and Crittenden (8). The saliva calibration solutions were chosen to approximate the ionic composition of saliva collected under stimulated conditions (8). These contained O-2 mM CaCl,, 50 mM NaCl, 5 mM KCl, 0.5 mM MgC12, and 10 mM Tris buffer (pH 7.4). Both the saliva and the serum, which was prepared by centrifuging blood which had clotted for 1 hr, were used without dilution. The luminescence was detected with a photomultiplier tube as previously described (3). In the experiments with the rapid mixing device, the photomultiplier tube was mounted at the top observation port. The photomultiplier tube current was recorded and photographed on a storage oscilloscope screen following current to voltage conversion or integration using the Keithley 602 in the ammeter or coulombmeter mode. RESULTS
Ah’D
DISCUSSION
The procedure of Shimomura et al. (1) was followed in the initial experiments (2). However, alteration of this procedure was necessary for theoretical and practical reasons. The theoretical criticism was that dilution of the serum samples and the addition of millimolar amounts of EDTA and Mg2+ to them will affect the equilibrium between ionized and nonionized calcium. In practice, it was found that if these procedures were not followed, the reaction rates were too fast to be measured accurately with the syringe and testtube mixing procedure. The result was poor reproducibility. Our more recent measurements were done with a Durrum (Model D110) stopped-flow rapid mixing device. Typical records are shown in Fig. 1. The degree of reproducibility can be appreciated by the fact that each of these traces are two different measurements superimposed. Figure 2 shows a typical calibration curve. Here, the logarithm of the maximum of the photomultiplier tube current has been plotted against
AFQUORIN-IONIC
CALCIUX
ASSAY
481
FIG. 1. Superimposed oscilloscope traces of the photomultiplier tube current as a function of time after mixing calcium-containing solutions with aequorin. The mixing was done in a Durrum stopped-flow rapid mixing device with a dead time of 3 msec. The traces from top to bottom were obtained with CaCh solutions of 2.0, 1.5, 1.0, and 0.5 mM, respectively. The final aequorin concentration was 0.06 mM, the highest achievable with the present purification procedure. Equal volumes of CaCI? and aequorin solutions were reacted. Each horizontal unit represents 10 msec.
0.5 Co hM1
1.0
1.5 2.0
FIG. 2. A typical calibration curve. The logarithms of maxima from current records such as those in Fig. 1 were plotted against the logarithms of the calcium concentrations of the test solutions. The results obtained with CaCL solutions are depicted by the solid circles. The open triangles represent the results obtained with CaCL solutions containing EDTA as described in the text.
482
IZUTSU
ET
AL.
the logarithm of the concentration of the CaCl, solutions used for calibration. The slope is indistinguishable from 2.0. In addition, the luminescence produced by solutions containing mixtures of calcium and EDTA (as well as NaCl) was measured and plotted on Fig. 2. All of these solutions had a calcium concentration of 2 mM with EDTA concentrations from 0 to 2.0 mM. The concentration of ionic calcium in these solutions was calculated by the method of Port,zehl et al. (10). These results are, in good agreement with the results obtained with CaCl, alone and indicate that, at least for these solutions, the system is sensitive to ionic calcium. The levels of ionic calcium in serum and saliva samples were estimated from their luminescence records using the appropriate calibration solutions. The results are given in Table 1. The average ionic calcium in the serum samples was about 1.75 mM. This value is appreciably higher than the values reported by others using the murexide (llJ2) procedure or the calcium electrode (13). Similarly, the ionic calcium in human parotid saliva as measured with aequorin (i.e., about 90% ionized) is significantly higher than the values obtained with murexide (14) and the calcium electrode (15). One possible explanation for this disparity is that the addition of the aequorin to the test solutions may disrupt their equilibrium. (The aequorin association constant for calcium is at least 7 X 10G M-l (16) ) . If this is the case, then one would expect the aequorin procedure to yield increasingly higher erroneous results when different chelaters with decreasing association constants are used. This was tested and confirmed by measuring the ionic calcium in solu-
Calcium
TABLE Concentrations
1 in Serum
Ionic
Saliva (1) (2) (3) (4)
calcium
Total
calciuma bM)
samples C.L. K.I. I.S.A. I.S.B.
(1) S.P.F. (2) K.I. (3) I.S. (4) D.W. (5) D.O.G. 5 Total
calcium bfif)
and Saliva
by atomic
absorption.
0.60 0.50 1.40 1.50
0.75 0.60 1.50 1.70
1.70 1.80 1.80 1.70 1.80
2.25 2.53 2.51 2.40 2.60
ADQUORIN-IOh-IC
CALCIUM
483
ASSAY
tions with total calcium concentrations of 2 mM but with increasing concentrations of EGTA, ATP, citrate, or phosphate. The results are given in Fig. 3. The results with EGTA were the same as with EDTA: The ionized calcium as measured with aequorin was in good agreement with the calculated value. For ATP, citrate, and phosphate, however, the aequorin procedure overestimated the ionic calcium concentrations in a manner consistent with the preceding discussion. If this explanation for the loss of accuracy in solutions containing sub,stances with low association constants is correct, then decreasing the aequorin concentration should decrease the error. Unfortunately, this procedure was nonproductive because dilution of the aequorin resulted in a marked decline in the slope of the calibration curve, rendering it useless. Dilution of the calcium samples, as suggested by Shimomura et al. (14)) was eliminated as a possibility because this would also disrupt their equilibrium. In conclusion, we note that the aequorin ionic calcium assay can 1000,
1’0 Awon
2‘0
3’0
concentrat!on
4’0
io
(mM)
FIG. 3. A comparisonof the ionic calcium concentrationsas measuredby the present aequorinprocedurewith the theoretically expected concentrationsin solutions containing one of five different calciumbinding substances. The ratios of the observedionic calciumconcentrationsto the theoretically expectedvalues were calculatedusingthe associationconstantsgiven in the referencescited after eachof the chelaters.The theoretical valueswere obtained by solving the simultaneousequations involving the equilibria of Ca*+and H’ with the various forms of the chelaters for a given pH and for given concentrationsof total calciumand total chelater.The abscissa gives the chelater concentrationin the test solutions.The open and closed circlesrepresentthe resultsobtained in the presenceof EGTA and EDTA respectively (10). The solid and open triangles represent the results with increasing concentrations of ATP (17) and citrate (X3), respectively, and the squares represent the results with phosphate(19). All solutions were at pH 7.4 and with 150 mM NaCl. Similar results were obtained at 25 and 5°C regardless of whether the maxima or initial slopes of the current records were used for calibration.
484
IZUTSU
ET
AL.
yield precise values for ionic calcium in test solutions which contain calcium chelaters with high association constants and also in test solutions without any calcium-binding agents. However, this assay requires a rapid mixing device since, at least under the present conditions, the halftime of rise is almost 4 msec (Fig. 1). A suitable method for use in the presence of chelaters with low association constants such as are found for the biological fluids including saliva and serum (20) has yet to be described. However, the present results indicate that the aequorin luminescence in biological systems containing components with high calcium association constants, e.g., sarcoplasmic reticulum, may be capable of precise calibration as suggested by Ashley (21). ACKNOWLEDGMENTS This study was supported by Public Health Service grant from the Washington State Sea Grant Program. Grant Program (c/o Division of Marine Resources, Seattle, 98195) also made the aequorin available to us.
grant DE-02609 and by a The Washington State Sea University of Washington,
REFEREECES 1. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1963) Science 140, 1339. 2. IZUTSU, K. T., AND FELTON, S. P. (1972) Clin. Chem. 18, 77. 3. IZUTSU, K. T., FELTON, S. P., SIEGEL, I. A., YODA, W. T., AND CHEN, A. C. X. (1972) Biochem. Biophys. Res. Commun. 49, 1034. 4. SHIMOMURA, O., AND JOHNSON, F. H. (1969) Biochemistry 8, 3991. 5. BAKER, P. F., HODGKIN, A. L., AND RIDGWAY, E. B. (1971). J. Physiol. 21,8, 709. 6. JOHNSON, F. H., AND SHIMOMURA, 0. (1972) Nature New Biol. 237, 287. 7. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1962) J. Cell. Comp. Physiol. 59, 223. 8. SCHNEYER, L. H., AND SCHNEYER, C. A., in Handbook of Physiology, Section 6: Alimentary Canal, Vol. 2. Secretion (Code, C. F., ed.), p. 497, Amer. Physiol. Sot., Washington, D.C., 1967. 9. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1963) J. Cell. Pomp. Physiol. 62, 1. 10. PORTZEHL, H., CALDWELL, P. C., AND RUEGG, J. C. (1964) Biochim. Biophys. Acta 79, 581. 11. WALSER, M. (1961) J. Clin. Invest. 40, 723. 12. ROSE, G. A. (1957) Clin. Chim. Acta 2, 227. 13. MOORE, E. W. (1970) J. Clin. Invest. 49, 318. 14. LIGHTFOOT, L., AND COOLIDGE, T. B. (1961) J. Dent. Res. 49, ~2. 15. GRON, P., SPINELLI, M. A., AND HAY, D. I., Program and Abstracts of Papers, 46th General Session, Int. Ass. Dent. Res., Vol. 58, 1968. 16. SHIMOMURA, O., AND JOHNSON, F. H. (1970) Nature 227, 1356. 17. NANNING.4, L. B. (1961) Biochim. Biophys. Acta 54, 330. 18. WALSER, M. (1961) J. Phys. Chem. 65, 159. 19. SMITH, R. M., AND ALBERTY, R. A. (1956) J. Amer. Chem. SOC. 78, 2376. 20. PEDERSON, B. 0. (1972) &and. J. Clin. Lab. Invest. 29, 75. 21. ASHLEY, C. C. (1970) J. Physiol. 219, 133P.
An
BIOCHEMISTRY
investigation on the
58,
479484
(1974)
of the
Ionic
Jellyfish
Protein
Calcium
for
,J. I. NICHOLLS, T. YODA
Research in Oral Biology and Anesthesiology Research University of Washington, Seattle, Washington Received
July
9, 1973;
accepted
Based
Aequorin
I<. T. IZUTSU, S. P. FELTON, I. A. SIEGEL, J. CRAWFORD, ,J. McGOUGH, AND W. Center
Assay
October
Center,
3, 1973
The ionic calcium assay based on the luminescence of the jellyfish protein aequorin was studied. A rapid mixing device was found to be necessary for good reproducibility. The accuracy of the procedure described could depend on the association constants of the calcium chelating agents involved in the test solutions. INTRODUCTION
Shimomura et al. (1) have described a method that may be capable of measuring the concentration of ionic calcium in serum. This procedure is based on the luminescence of the jellyfish protein aequorin. Our previous attempts (2) to utilize this procedure were only partially successful because of the difficulty in achieving rapid, consistent mixing and because the serum samples were diluted to slow the reaction to a measurable rate. We report here on our further studies of this assay. In particular, we have found that a rapid mixing device is necessary for consistent results and that the accuracy of the assay depends on the association constant of the calcium buffer used in the test solution. METHODS
The aequorin was prepared as descri,bed elsewhere (2,3) except that the sephadex column chromatography was done at pH 7.5. The specific activity of the aequorin used in these studies was approximately 1.5 X 1Ol5 photons/mg protein. This is a third of the value reported by Shimomura and Johnson (4) for pure aequorin and about eight times higher than the value reported by Baker et al. (5) for the aequorin used in their physiological studies. This specific activity is also approximately the same as the value reported by Shimomura et al. (1) for the aequorin used in their serum studies and also within the range of specific activities given by Johnson and Shimomura (6) as being adequate for most purposes. Further chromatography (on DEAE) resulted in a higher Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
479 Inc. reserved.
480
IZUTSU
ET
.4L.
specific activity, but this was offset by a decreased yield which made the procedure impractical when large amounts of aequorin are necessary as has been previously recognized (5,6). The major contaminant of our aequorin was probably the green protein (7) since there was a detectable 510 nm fluorescence with 460 excitation. The primary calibration solutions contained between 0 and 3 mM CaCl, as well as 150 mM NaCl. The addition of 10 mM Tris buffer (pH 7.4) to these solutions had no effect on the calibration curves. The serum calibration standards contained 0.75 mM MgCl*, 10 mM Tris buffer (pH 7.4)) and 5 mM KC1 as well as NaCl and CaCl, since magnesium is known to affect the rate of the aequorin-calcium reaction (7). Saliva was collected under stimulated conditions (while sucking sour lemon drops) by the method of Carlson and Crittenden (8). The saliva calibration solutions were chosen to approximate the ionic composition of saliva collected under stimulated conditions (8). These contained O-2 mM CaCl,, 50 mM NaCl, 5 mM KCl, 0.5 mM MgC12, and 10 mM Tris buffer (pH 7.4). Both the saliva and the serum, which was prepared by centrifuging blood which had clotted for 1 hr, were used without dilution. The luminescence was detected with a photomultiplier tube as previously described (3). In the experiments with the rapid mixing device, the photomultiplier tube was mounted at the top observation port. The photomultiplier tube current was recorded and photographed on a storage oscilloscope screen following current to voltage conversion or integration using the Keithley 602 in the ammeter or coulombmeter mode. RESULTS
Ah’D
DISCUSSION
The procedure of Shimomura et al. (1) was followed in the initial experiments (2). However, alteration of this procedure was necessary for theoretical and practical reasons. The theoretical criticism was that dilution of the serum samples and the addition of millimolar amounts of EDTA and Mg2+ to them will affect the equilibrium between ionized and nonionized calcium. In practice, it was found that if these procedures were not followed, the reaction rates were too fast to be measured accurately with the syringe and testtube mixing procedure. The result was poor reproducibility. Our more recent measurements were done with a Durrum (Model D110) stopped-flow rapid mixing device. Typical records are shown in Fig. 1. The degree of reproducibility can be appreciated by the fact that each of these traces are two different measurements superimposed. Figure 2 shows a typical calibration curve. Here, the logarithm of the maximum of the photomultiplier tube current has been plotted against
AFQUORIN-IONIC
CALCIUX
ASSAY
481
FIG. 1. Superimposed oscilloscope traces of the photomultiplier tube current as a function of time after mixing calcium-containing solutions with aequorin. The mixing was done in a Durrum stopped-flow rapid mixing device with a dead time of 3 msec. The traces from top to bottom were obtained with CaCh solutions of 2.0, 1.5, 1.0, and 0.5 mM, respectively. The final aequorin concentration was 0.06 mM, the highest achievable with the present purification procedure. Equal volumes of CaCI? and aequorin solutions were reacted. Each horizontal unit represents 10 msec.
0.5 Co hM1
1.0
1.5 2.0
FIG. 2. A typical calibration curve. The logarithms of maxima from current records such as those in Fig. 1 were plotted against the logarithms of the calcium concentrations of the test solutions. The results obtained with CaCL solutions are depicted by the solid circles. The open triangles represent the results obtained with CaCL solutions containing EDTA as described in the text.
482
IZUTSU
ET
AL.
the logarithm of the concentration of the CaCl, solutions used for calibration. The slope is indistinguishable from 2.0. In addition, the luminescence produced by solutions containing mixtures of calcium and EDTA (as well as NaCl) was measured and plotted on Fig. 2. All of these solutions had a calcium concentration of 2 mM with EDTA concentrations from 0 to 2.0 mM. The concentration of ionic calcium in these solutions was calculated by the method of Port,zehl et al. (10). These results are, in good agreement with the results obtained with CaCl, alone and indicate that, at least for these solutions, the system is sensitive to ionic calcium. The levels of ionic calcium in serum and saliva samples were estimated from their luminescence records using the appropriate calibration solutions. The results are given in Table 1. The average ionic calcium in the serum samples was about 1.75 mM. This value is appreciably higher than the values reported by others using the murexide (llJ2) procedure or the calcium electrode (13). Similarly, the ionic calcium in human parotid saliva as measured with aequorin (i.e., about 90% ionized) is significantly higher than the values obtained with murexide (14) and the calcium electrode (15). One possible explanation for this disparity is that the addition of the aequorin to the test solutions may disrupt their equilibrium. (The aequorin association constant for calcium is at least 7 X 10G M-l (16) ) . If this is the case, then one would expect the aequorin procedure to yield increasingly higher erroneous results when different chelaters with decreasing association constants are used. This was tested and confirmed by measuring the ionic calcium in solu-
Calcium
TABLE Concentrations
1 in Serum
Ionic
Saliva (1) (2) (3) (4)
calcium
Total
calciuma bM)
samples C.L. K.I. I.S.A. I.S.B.
(1) S.P.F. (2) K.I. (3) I.S. (4) D.W. (5) D.O.G. 5 Total
calcium bfif)
and Saliva
by atomic
absorption.
0.60 0.50 1.40 1.50
0.75 0.60 1.50 1.70
1.70 1.80 1.80 1.70 1.80
2.25 2.53 2.51 2.40 2.60
ADQUORIN-IOh-IC
CALCIUM
483
ASSAY
tions with total calcium concentrations of 2 mM but with increasing concentrations of EGTA, ATP, citrate, or phosphate. The results are given in Fig. 3. The results with EGTA were the same as with EDTA: The ionized calcium as measured with aequorin was in good agreement with the calculated value. For ATP, citrate, and phosphate, however, the aequorin procedure overestimated the ionic calcium concentrations in a manner consistent with the preceding discussion. If this explanation for the loss of accuracy in solutions containing sub,stances with low association constants is correct, then decreasing the aequorin concentration should decrease the error. Unfortunately, this procedure was nonproductive because dilution of the aequorin resulted in a marked decline in the slope of the calibration curve, rendering it useless. Dilution of the calcium samples, as suggested by Shimomura et al. (14)) was eliminated as a possibility because this would also disrupt their equilibrium. In conclusion, we note that the aequorin ionic calcium assay can 1000,
1’0 Awon
2‘0
3’0
concentrat!on
4’0
io
(mM)
FIG. 3. A comparisonof the ionic calcium concentrationsas measuredby the present aequorinprocedurewith the theoretically expected concentrationsin solutions containing one of five different calciumbinding substances. The ratios of the observedionic calciumconcentrationsto the theoretically expectedvalues were calculatedusingthe associationconstantsgiven in the referencescited after eachof the chelaters.The theoretical valueswere obtained by solving the simultaneousequations involving the equilibria of Ca*+and H’ with the various forms of the chelaters for a given pH and for given concentrationsof total calciumand total chelater.The abscissa gives the chelater concentrationin the test solutions.The open and closed circlesrepresentthe resultsobtained in the presenceof EGTA and EDTA respectively (10). The solid and open triangles represent the results with increasing concentrations of ATP (17) and citrate (X3), respectively, and the squares represent the results with phosphate(19). All solutions were at pH 7.4 and with 150 mM NaCl. Similar results were obtained at 25 and 5°C regardless of whether the maxima or initial slopes of the current records were used for calibration.
484
IZUTSU
ET
AL.
yield precise values for ionic calcium in test solutions which contain calcium chelaters with high association constants and also in test solutions without any calcium-binding agents. However, this assay requires a rapid mixing device since, at least under the present conditions, the halftime of rise is almost 4 msec (Fig. 1). A suitable method for use in the presence of chelaters with low association constants such as are found for the biological fluids including saliva and serum (20) has yet to be described. However, the present results indicate that the aequorin luminescence in biological systems containing components with high calcium association constants, e.g., sarcoplasmic reticulum, may be capable of precise calibration as suggested by Ashley (21). ACKNOWLEDGMENTS This study was supported by Public Health Service grant from the Washington State Sea Grant Program. Grant Program (c/o Division of Marine Resources, Seattle, 98195) also made the aequorin available to us.
grant DE-02609 and by a The Washington State Sea University of Washington,
REFEREECES 1. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1963) Science 140, 1339. 2. IZUTSU, K. T., AND FELTON, S. P. (1972) Clin. Chem. 18, 77. 3. IZUTSU, K. T., FELTON, S. P., SIEGEL, I. A., YODA, W. T., AND CHEN, A. C. X. (1972) Biochem. Biophys. Res. Commun. 49, 1034. 4. SHIMOMURA, O., AND JOHNSON, F. H. (1969) Biochemistry 8, 3991. 5. BAKER, P. F., HODGKIN, A. L., AND RIDGWAY, E. B. (1971). J. Physiol. 21,8, 709. 6. JOHNSON, F. H., AND SHIMOMURA, 0. (1972) Nature New Biol. 237, 287. 7. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1962) J. Cell. Comp. Physiol. 59, 223. 8. SCHNEYER, L. H., AND SCHNEYER, C. A., in Handbook of Physiology, Section 6: Alimentary Canal, Vol. 2. Secretion (Code, C. F., ed.), p. 497, Amer. Physiol. Sot., Washington, D.C., 1967. 9. SHIMOMURA, O., JOHNSON, F. H., AND SAIGA, Y. (1963) J. Cell. Pomp. Physiol. 62, 1. 10. PORTZEHL, H., CALDWELL, P. C., AND RUEGG, J. C. (1964) Biochim. Biophys. Acta 79, 581. 11. WALSER, M. (1961) J. Clin. Invest. 40, 723. 12. ROSE, G. A. (1957) Clin. Chim. Acta 2, 227. 13. MOORE, E. W. (1970) J. Clin. Invest. 49, 318. 14. LIGHTFOOT, L., AND COOLIDGE, T. B. (1961) J. Dent. Res. 49, ~2. 15. GRON, P., SPINELLI, M. A., AND HAY, D. I., Program and Abstracts of Papers, 46th General Session, Int. Ass. Dent. Res., Vol. 58, 1968. 16. SHIMOMURA, O., AND JOHNSON, F. H. (1970) Nature 227, 1356. 17. NANNING.4, L. B. (1961) Biochim. Biophys. Acta 54, 330. 18. WALSER, M. (1961) J. Phys. Chem. 65, 159. 19. SMITH, R. M., AND ALBERTY, R. A. (1956) J. Amer. Chem. SOC. 78, 2376. 20. PEDERSON, B. 0. (1972) &and. J. Clin. Lab. Invest. 29, 75. 21. ASHLEY, C. C. (1970) J. Physiol. 219, 133P.