ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 632-634, 1979
COMMUNICATIONS Affinity
of Folic Acid for the Folate-Binding
Protein of Choroid Plexus
The affmity of folic acid for the folate-binding protein of rabbit choroid plexus was determined by equilibrium dialysis at 4°C. All solutions contained 0.02% Triton X-100 to maintain the binder in solution. At pH 7.0, the apparent dissociation constant (K,) at a binder concentration of 0.36 nM was 9.4 pM with slight positive cooperativity (Hill coefficient = 1.19). The K, increased at pH 6.0 and when a higher concentration of binder (3.25 nM) was used to 30.1 and 46.0 PM, respectively. However, the maximal binding capacity per milligram of protein did notchange. At pH 5.0, the K, was greater than 20 nM. These results show that the affinity of the choroid plexus folate-binding protein (when solubilized in Triton X-100) for folic acid depends on both the concentration of binder and the pH. The choroid plexus, the anatomical locus of the blood cerebrospinal fluid (CSF)’ barrier, contains a highaffinity folate-binding protein localized, in large part, within the cellular plasma membranes (1, 2). This folate binding protein (binder) is involved in the transfer of folates between blood and CSF through the choroid plexus (1, 2). Recently, the folate binder of choroid plexus was solubilized in Triton X-100, partially purified, and characterized (1). Working in 0.02% Triton X-100 to maintain the binder in solution, the dissociation constant (K,J of folate for the binder was less than 10e8 M (1) at pH 7.0. The Kd at lower pH values increased (1). Kinetic studies revealed the rate of dissociation of the binder-folic acid complex (1 nM) to be less than 5% in 2 h. The purpose of the present study was to determine the afflnity of folate for the folate-binding protein of choroid plexus at several pH values and concentrations of binder using equilibrium dialysis. A practical method was developed to accelerate the attainment of equilibrium.
Methods The choroid plexus folate binder (in homogenates) was freed from endogenous folates and solubilized in Triton X-100 by methods previously described in detail (1). Briefly, fresh rabbit choroid plexus (about 50 mg) was homogenized and incubated for 2 h in 1 ml 2% Triton X-100 (1). Then, the homogenate was acidified to pH 2.8 with 1 M citric acid. After 45 min at l”C, 0.2 ml of a charcoaValbumin suspension was added to the acidified homogenate to separate the binder from the folates (1). After 30 min at l”C, the mixture was centrifuged, 1 ml supernatant was withdrawn, and the pH was adjusted to 7.0 with NaOH (1). The endogenous folates, associated with the charcoal pellet, were thus separated from the folate binder which remained in the supernatant. The various preparations contained 16 to 1 Abbreviation
used: CSF, cerebrospinal fluid.
0003-9861/79/060632-03$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
21 pmol of folate-binding ability per milligram of protein (1). Equilibrium dialysis was performed using Spectrapor 1 dialysis tubing (Fisher Scientific) in 0.01 M sodium phosphate buffer (pH 6.0 or ‘7.0). Various amounts of the choroid plexus folate-binding protein (preparation) were dissolved in the internal solution (1.0 ml inside the dialysis bag). [3H]Folic acid was added to the internal and/or external solution (30 ml). The outer solution was stirred with a 2-cm Teflon stirring bar at 100 rpm. All dialysis solutions contained 0.02% Triton X-100 (to keep the binder in solution) and sodium azide (to inhibit bacterial growth). All dialysis experiments were carried out at 4°C. [3H]Folic acid (Amersham/Searle, Inc., 45 Ci/mmol) was purified biweekly. The binding data (at equilibrium) were analyzed using the general theory of multiple equilibria with the assumption of identical sites with interaction between them (3). The Hill plot was employed for graphical purposes (4). Comparable data points (on Hill plots) were fitted using linear regression analysis and the method of least squares (5). Comparison of regression lines for identity and slope, as well as 95% confidence limits for intercepts and slopes, were calculated using standard parametric statistical methods (5). Scintillation spectrometry and protein determinations in the presence of Triton X-100 were performed as previously described (1).
Results When sufficient [3H]folic acid was added to either the inside or outside of the dialysis bags to give an equilibrium concentration of 2 pmol/ml, equilibrium was attained (at pH 6.0 or 7.0) within 24 h if no binder was added to the dialysis bag. However, if only 5% as much [3H]folic acid was added, equilibrium was not achieved by 24 h and required 40 h. When the folate binder was added to the dialysis bag and the [3H]folic acid was added initially to either the inside or outside of the dialysis bag, equilibrium was not attained even after ‘7 days under some conditions. 632
AFFINITY
OF FOLATE
FOR CHOROID PLEXUS
FOLATE
BINDER
633
gests that there may be a slight positive cooperativity to the binding (4). At pH 6.0 and 5.0, as expected, the K,, increased significantly. However, the maximal binding capacity per milligram of protein at pH 6.0 was not significantly different than at pH 7.0. The K,, increased by five times at pH 7.0 when the amount of binder was increased from 0.36 to 3.25 pmol/ml inside the dialysis bag (Table I). How-ever, the maximal binding capacity per milligram of protein did not change at this concentration of binder.
Discussion FIG. 1. Binding of [3H]folic acid (pmoYmg protein) inside the dialysis bag (A) and concentration of [3H]folic acid outside of the dialysis bag(B) as a function of time. Choroid plexus protein, 76 pg, containing the folate binder (able to bind 20.2 pmol folic acid/mg protein) was added to 1.0 ml of phosphate buffer (pH 7.0). Then 0.88 pmol [3H]folic acid was added, and the mixture was incubated at 4°C for 5 min, transferred to a dialysis bag, and dialyzed for 7 days. At various times, duplicate aliquots of the external solution were assayed for [SH]folic acid (curve 1, B). With the assumption that the [3H]folic acid concentration outside the bag equaled the concentration of unbound [3H]folic acid inside the bag at all times, the bound [3H]folic acid inside the bag was calculated by subtraction (curve 1, A). Also, after 7 days, the [3H]folic acid within the bag was assayed directly. Similar experiments were done except that all of the [3H]folic acid was placed outside the bag (curve 2), or 0.22 pmol [3H]folic acid was placed outside the bag and 0.66 pmol [3H]folic acid was placed inside the bag at the start of the dialysis (curve 3).
A representative experiment is shown in Fig. 1 (curves 1 and 2). To accelerate the attainment of equilibrium in subsequent experiments, the approximate amounts (previously determined by trial and error) of [3H]folic acid at equilibrium were added to the inside and outside of the dialysis bag at the start of the dialysis (curve 3, Fig. 1). In all subsequent equilibrium dialysis experiments described, data points were considered valid only if there was less than a 10% change in the total concentration of [3H]folic acid inside and outside the dialysis bags after 3 or 4 days of dialysis, i.e., the concentrations of [3H]folic acid inside and outside of the dialysis bag at the start of the dialysis were about the equilibrium concentrations. Shown in Fig. 2 are Hill plots of the equilibrium dialysis data at pH 6.0 and 7.0. The Hill coefficient and apparent dissociation constant (K,) derived from the Hill plots are shown in Table I. The slope of the line in Fig. 1, i.e., the Hill coefficient at pH 6.0 (0.90), was significantly different from the Hill coefficient at pH 7.0 (1.19) (Table I). This “nonhyperbolicity” at pH 7.0 sug-
The results reported herein (Table I) confirm the existence of a very high-affinity folate-binding macromolecule in rabbit choroid plexus by an independent method (equilibrium dialysis) (1, 2). The method employed (i.e., adding the equilibrium concentrations of folic acid) circumvented the slowness of attaining equilibrium when the folic acid was added exclusively to either the outside or inside of the dialysis bag (Fig. 1). The exact reason(s) for the extremely slow attainment of equilibrium (Fig. 1) is unclear but could be related to the tightness of the binding, the presence of Triton X-100 in free and micellular forms in the solution, and/or self-association (e.g., “ring stacking”) of folates, although unlikely (see below) (6, 7). Biweekly purification of the [3H]folic acid was necessary to avoid 3H not associated with the folic acid (1).
log
F
FIG. 2. Plot of log [e/(1 - 0)] versus the log of the unbound folate concentration (F) (Hill plot) (4, 8, 9) at pH 6.0 and 7.0. 0 is the percentage of maximal folate binding at equilibrium at a given unbound folate concentration (pmopml). Maximal folate binding was determined by equilibrium dialysis.
634
REYNOLD
SPECTOR
TABLE
I
SLOPE (HILL COEFFICIENT) AND APPARENT DISSOCIATION CONSTANT (K,) OF FOLIC ACID FOR THE CHOROID PLEXUS FOLATE-BINDING PROTEIN UNDER VARIOUS CONDITIONS” Condition Concentration of binder (pmoYml)
PH
Hill coefficient
0.36 0.43 3.25
7.0 6.0 7.0
0.90 + 0.13 (20)”
30.1 (15.3-59.2)”
1.29 k 0.18 (10)
0.70
5.0
46.0 (21.7-100.1) >20 x 10YC
K, (PM)
1.19 + 0.17 (11)
9.4 (4.8-18.6)
” Indicated for the Hill coefficients and the K, are the means and the 95% confidence limits. The number of points at various concentrations of binder are indicated in parentheses beside the Hill coefficients. Each point is the mean of two determinations. h The Hill coefficient at pH 6.0 is significantly different (P < 0.05) from the Hill coefficient at pH 7.0. c Values of K,, are significantly different (P < 0.05) from the K, (=9.4) determined at pH 7.0 with 0.36 pmoliml binder in the medium. The binding properties of the solubilized membranebound choroid plexus folate binder are, in some respects, similar to those of several water-soluble folate binders (8 11) but differ from the weak folate binders in serum which have a K,, for folate of 1.0 mM (12). For example, the very low apparent dissociation constant (9 PM) (Table I) at pH 7.0 is comparable to the K, reported for dilute solutions of the water-soluble folate binders in milk and hog kidney (8- 11). As previously reported for the choroid plexus binder (1) and other folate binders (8-lo), the K, increases with decreasing pH (Table I). Like the folate-binding protein in milk at pH 7.0 (8-lo), the interaction between the folate and the binder may not be hyperbolic as demonstrated by Hill plots (Fig. 1, Table 1). The explanation for the presumed positive cooperativity is unclear (4, 6, 7). Moreover, the K, increases with an increase in the concentration of binder (Table I). This phenomenon has been described with the water-soluble folate binder in milk (8-10). However, the binding capacity per milligram of protein did not change unlike the folate binder in milk (8-10). In summary, the choroid plexus contains a folatebinding protein (l), which, when solubilized in 0.02% Triton X-100, has a very high affinity for folic acid at neutral pH. ACKNOWLEDGMENTS
The expert technical assistance of Paul Kelley is acknowledged. This work was supported in part by grants from the NIH (NS 14211) and the National Foundation-March of Dimes. Dr. Spector is the Recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association.
REFERENCES
1. SPECTOR, R. (1977) J. Biol. Chem. 252, 33643370. 2. SPECTOR, R. (1977) Bruin Res. 134, 573-576. 3. TANFORD, C. (1967) Physical Chemistry of Macromolecules, pp. 526-548, Wiley, New York. 4. GAKLER, E. (1977) Phmmazie 12, 739-746. 5. PASTORE, E. J. (1971)Ann. N. Y. Acad. Sci. 186, 43-54. 6. PASTORE, E. J., WRIGHT, J. M., AND KAPLAN, N. 0. (1976) Fed. Proc. 35, 538. 7. BROWNLEE, K. A. (1965) Statistical Theory and Methodology, 2nd ed., pp. 349-361, Wiley, New York. 8. INGEMANN HANSEN, S., HOLM, J., AND LYNGBYE, J. (1977) Stand. J. Clin. Lab. Med. 37, 363-367. 9. HOLM, J., INGEMANN HANSEN, S., AND LYNG BYE, J. (1978) Acta Phmmmol. Toxicol. 42, 77-80. 10. INGEMANN HANSEN, S., HOLM, J., AND LYNG BYE, J. (1977) Clin. Chim. Acta 75, 321-324. 11. WAXMAN, S., SCHEIBER, C., AND RUBINOFF, M. (1977) in Advances in Nutritional Research (Draper, E. H., ed.), Vol. 1, pp. 55-76, Plenum, New York. 12. ZE~ER, A., AND DULY, P. E. (1978) Ann. Clin. Lab.
Sci. 8, 57-63.
REYNOW SPECTOR Departments University Zowa City, Received
of Medicine and Pharmacology of Iowa College of Medicine Iowa 52242 August 25, 1978; revised February
12, 1979
COMMUNICATIONS Affinity
of Folic Acid for the Folate-Binding
Protein of Choroid Plexus
The affmity of folic acid for the folate-binding protein of rabbit choroid plexus was determined by equilibrium dialysis at 4°C. All solutions contained 0.02% Triton X-100 to maintain the binder in solution. At pH 7.0, the apparent dissociation constant (K,) at a binder concentration of 0.36 nM was 9.4 pM with slight positive cooperativity (Hill coefficient = 1.19). The K, increased at pH 6.0 and when a higher concentration of binder (3.25 nM) was used to 30.1 and 46.0 PM, respectively. However, the maximal binding capacity per milligram of protein did notchange. At pH 5.0, the K, was greater than 20 nM. These results show that the affinity of the choroid plexus folate-binding protein (when solubilized in Triton X-100) for folic acid depends on both the concentration of binder and the pH. The choroid plexus, the anatomical locus of the blood cerebrospinal fluid (CSF)’ barrier, contains a highaffinity folate-binding protein localized, in large part, within the cellular plasma membranes (1, 2). This folate binding protein (binder) is involved in the transfer of folates between blood and CSF through the choroid plexus (1, 2). Recently, the folate binder of choroid plexus was solubilized in Triton X-100, partially purified, and characterized (1). Working in 0.02% Triton X-100 to maintain the binder in solution, the dissociation constant (K,J of folate for the binder was less than 10e8 M (1) at pH 7.0. The Kd at lower pH values increased (1). Kinetic studies revealed the rate of dissociation of the binder-folic acid complex (1 nM) to be less than 5% in 2 h. The purpose of the present study was to determine the afflnity of folate for the folate-binding protein of choroid plexus at several pH values and concentrations of binder using equilibrium dialysis. A practical method was developed to accelerate the attainment of equilibrium.
Methods The choroid plexus folate binder (in homogenates) was freed from endogenous folates and solubilized in Triton X-100 by methods previously described in detail (1). Briefly, fresh rabbit choroid plexus (about 50 mg) was homogenized and incubated for 2 h in 1 ml 2% Triton X-100 (1). Then, the homogenate was acidified to pH 2.8 with 1 M citric acid. After 45 min at l”C, 0.2 ml of a charcoaValbumin suspension was added to the acidified homogenate to separate the binder from the folates (1). After 30 min at l”C, the mixture was centrifuged, 1 ml supernatant was withdrawn, and the pH was adjusted to 7.0 with NaOH (1). The endogenous folates, associated with the charcoal pellet, were thus separated from the folate binder which remained in the supernatant. The various preparations contained 16 to 1 Abbreviation
used: CSF, cerebrospinal fluid.
0003-9861/79/060632-03$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
21 pmol of folate-binding ability per milligram of protein (1). Equilibrium dialysis was performed using Spectrapor 1 dialysis tubing (Fisher Scientific) in 0.01 M sodium phosphate buffer (pH 6.0 or ‘7.0). Various amounts of the choroid plexus folate-binding protein (preparation) were dissolved in the internal solution (1.0 ml inside the dialysis bag). [3H]Folic acid was added to the internal and/or external solution (30 ml). The outer solution was stirred with a 2-cm Teflon stirring bar at 100 rpm. All dialysis solutions contained 0.02% Triton X-100 (to keep the binder in solution) and sodium azide (to inhibit bacterial growth). All dialysis experiments were carried out at 4°C. [3H]Folic acid (Amersham/Searle, Inc., 45 Ci/mmol) was purified biweekly. The binding data (at equilibrium) were analyzed using the general theory of multiple equilibria with the assumption of identical sites with interaction between them (3). The Hill plot was employed for graphical purposes (4). Comparable data points (on Hill plots) were fitted using linear regression analysis and the method of least squares (5). Comparison of regression lines for identity and slope, as well as 95% confidence limits for intercepts and slopes, were calculated using standard parametric statistical methods (5). Scintillation spectrometry and protein determinations in the presence of Triton X-100 were performed as previously described (1).
Results When sufficient [3H]folic acid was added to either the inside or outside of the dialysis bags to give an equilibrium concentration of 2 pmol/ml, equilibrium was attained (at pH 6.0 or 7.0) within 24 h if no binder was added to the dialysis bag. However, if only 5% as much [3H]folic acid was added, equilibrium was not achieved by 24 h and required 40 h. When the folate binder was added to the dialysis bag and the [3H]folic acid was added initially to either the inside or outside of the dialysis bag, equilibrium was not attained even after ‘7 days under some conditions. 632
AFFINITY
OF FOLATE
FOR CHOROID PLEXUS
FOLATE
BINDER
633
gests that there may be a slight positive cooperativity to the binding (4). At pH 6.0 and 5.0, as expected, the K,, increased significantly. However, the maximal binding capacity per milligram of protein at pH 6.0 was not significantly different than at pH 7.0. The K,, increased by five times at pH 7.0 when the amount of binder was increased from 0.36 to 3.25 pmol/ml inside the dialysis bag (Table I). How-ever, the maximal binding capacity per milligram of protein did not change at this concentration of binder.
Discussion FIG. 1. Binding of [3H]folic acid (pmoYmg protein) inside the dialysis bag (A) and concentration of [3H]folic acid outside of the dialysis bag(B) as a function of time. Choroid plexus protein, 76 pg, containing the folate binder (able to bind 20.2 pmol folic acid/mg protein) was added to 1.0 ml of phosphate buffer (pH 7.0). Then 0.88 pmol [3H]folic acid was added, and the mixture was incubated at 4°C for 5 min, transferred to a dialysis bag, and dialyzed for 7 days. At various times, duplicate aliquots of the external solution were assayed for [SH]folic acid (curve 1, B). With the assumption that the [3H]folic acid concentration outside the bag equaled the concentration of unbound [3H]folic acid inside the bag at all times, the bound [3H]folic acid inside the bag was calculated by subtraction (curve 1, A). Also, after 7 days, the [3H]folic acid within the bag was assayed directly. Similar experiments were done except that all of the [3H]folic acid was placed outside the bag (curve 2), or 0.22 pmol [3H]folic acid was placed outside the bag and 0.66 pmol [3H]folic acid was placed inside the bag at the start of the dialysis (curve 3).
A representative experiment is shown in Fig. 1 (curves 1 and 2). To accelerate the attainment of equilibrium in subsequent experiments, the approximate amounts (previously determined by trial and error) of [3H]folic acid at equilibrium were added to the inside and outside of the dialysis bag at the start of the dialysis (curve 3, Fig. 1). In all subsequent equilibrium dialysis experiments described, data points were considered valid only if there was less than a 10% change in the total concentration of [3H]folic acid inside and outside the dialysis bags after 3 or 4 days of dialysis, i.e., the concentrations of [3H]folic acid inside and outside of the dialysis bag at the start of the dialysis were about the equilibrium concentrations. Shown in Fig. 2 are Hill plots of the equilibrium dialysis data at pH 6.0 and 7.0. The Hill coefficient and apparent dissociation constant (K,) derived from the Hill plots are shown in Table I. The slope of the line in Fig. 1, i.e., the Hill coefficient at pH 6.0 (0.90), was significantly different from the Hill coefficient at pH 7.0 (1.19) (Table I). This “nonhyperbolicity” at pH 7.0 sug-
The results reported herein (Table I) confirm the existence of a very high-affinity folate-binding macromolecule in rabbit choroid plexus by an independent method (equilibrium dialysis) (1, 2). The method employed (i.e., adding the equilibrium concentrations of folic acid) circumvented the slowness of attaining equilibrium when the folic acid was added exclusively to either the outside or inside of the dialysis bag (Fig. 1). The exact reason(s) for the extremely slow attainment of equilibrium (Fig. 1) is unclear but could be related to the tightness of the binding, the presence of Triton X-100 in free and micellular forms in the solution, and/or self-association (e.g., “ring stacking”) of folates, although unlikely (see below) (6, 7). Biweekly purification of the [3H]folic acid was necessary to avoid 3H not associated with the folic acid (1).
log
F
FIG. 2. Plot of log [e/(1 - 0)] versus the log of the unbound folate concentration (F) (Hill plot) (4, 8, 9) at pH 6.0 and 7.0. 0 is the percentage of maximal folate binding at equilibrium at a given unbound folate concentration (pmopml). Maximal folate binding was determined by equilibrium dialysis.
634
REYNOLD
SPECTOR
TABLE
I
SLOPE (HILL COEFFICIENT) AND APPARENT DISSOCIATION CONSTANT (K,) OF FOLIC ACID FOR THE CHOROID PLEXUS FOLATE-BINDING PROTEIN UNDER VARIOUS CONDITIONS” Condition Concentration of binder (pmoYml)
PH
Hill coefficient
0.36 0.43 3.25
7.0 6.0 7.0
0.90 + 0.13 (20)”
30.1 (15.3-59.2)”
1.29 k 0.18 (10)
0.70
5.0
46.0 (21.7-100.1) >20 x 10YC
K, (PM)
1.19 + 0.17 (11)
9.4 (4.8-18.6)
” Indicated for the Hill coefficients and the K, are the means and the 95% confidence limits. The number of points at various concentrations of binder are indicated in parentheses beside the Hill coefficients. Each point is the mean of two determinations. h The Hill coefficient at pH 6.0 is significantly different (P < 0.05) from the Hill coefficient at pH 7.0. c Values of K,, are significantly different (P < 0.05) from the K, (=9.4) determined at pH 7.0 with 0.36 pmoliml binder in the medium. The binding properties of the solubilized membranebound choroid plexus folate binder are, in some respects, similar to those of several water-soluble folate binders (8 11) but differ from the weak folate binders in serum which have a K,, for folate of 1.0 mM (12). For example, the very low apparent dissociation constant (9 PM) (Table I) at pH 7.0 is comparable to the K, reported for dilute solutions of the water-soluble folate binders in milk and hog kidney (8- 11). As previously reported for the choroid plexus binder (1) and other folate binders (8-lo), the K, increases with decreasing pH (Table I). Like the folate-binding protein in milk at pH 7.0 (8-lo), the interaction between the folate and the binder may not be hyperbolic as demonstrated by Hill plots (Fig. 1, Table 1). The explanation for the presumed positive cooperativity is unclear (4, 6, 7). Moreover, the K, increases with an increase in the concentration of binder (Table I). This phenomenon has been described with the water-soluble folate binder in milk (8-10). However, the binding capacity per milligram of protein did not change unlike the folate binder in milk (8-10). In summary, the choroid plexus contains a folatebinding protein (l), which, when solubilized in 0.02% Triton X-100, has a very high affinity for folic acid at neutral pH. ACKNOWLEDGMENTS
The expert technical assistance of Paul Kelley is acknowledged. This work was supported in part by grants from the NIH (NS 14211) and the National Foundation-March of Dimes. Dr. Spector is the Recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association.
REFERENCES
1. SPECTOR, R. (1977) J. Biol. Chem. 252, 33643370. 2. SPECTOR, R. (1977) Bruin Res. 134, 573-576. 3. TANFORD, C. (1967) Physical Chemistry of Macromolecules, pp. 526-548, Wiley, New York. 4. GAKLER, E. (1977) Phmmazie 12, 739-746. 5. PASTORE, E. J. (1971)Ann. N. Y. Acad. Sci. 186, 43-54. 6. PASTORE, E. J., WRIGHT, J. M., AND KAPLAN, N. 0. (1976) Fed. Proc. 35, 538. 7. BROWNLEE, K. A. (1965) Statistical Theory and Methodology, 2nd ed., pp. 349-361, Wiley, New York. 8. INGEMANN HANSEN, S., HOLM, J., AND LYNGBYE, J. (1977) Stand. J. Clin. Lab. Med. 37, 363-367. 9. HOLM, J., INGEMANN HANSEN, S., AND LYNG BYE, J. (1978) Acta Phmmmol. Toxicol. 42, 77-80. 10. INGEMANN HANSEN, S., HOLM, J., AND LYNG BYE, J. (1977) Clin. Chim. Acta 75, 321-324. 11. WAXMAN, S., SCHEIBER, C., AND RUBINOFF, M. (1977) in Advances in Nutritional Research (Draper, E. H., ed.), Vol. 1, pp. 55-76, Plenum, New York. 12. ZE~ER, A., AND DULY, P. E. (1978) Ann. Clin. Lab.
Sci. 8, 57-63.
REYNOW SPECTOR Departments University Zowa City, Received
of Medicine and Pharmacology of Iowa College of Medicine Iowa 52242 August 25, 1978; revised February
12, 1979
