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Studies on the kinetics of reaction and hydrolysis of fluorescamine
ARCHIVES
Studies
OF BIOCHEMISTRY
AND
BIOPHYSICS
on the Kinetics STANLEY
STEIN, Roche Institute
163, 400-403 (1974)
of Reaction PETER
and Hydrolysis
BiZHLEN,
of Molecular
Biology,
Received January
AND
SIDNEY
Nutley,
of Fluorescamine UDENFRIEND
New Jersey 07110
28, 1974
The influence of various parameters on the rate of reaction of fluorescamine with primary amines and on the rate of hydrolysis of the reagent is described. The studies indicate that both are dependent on the reaction conditions, includingpH, solvent in which the reagent is prepared, temperature, reagent concentration, and buffer salt. Under any set of conditions the reaction rates vary with the amines. A correlation between reaction rate and extent of fluorophor formation has been demonstrated. Kinetic evidence for a multistep reaction mechanism, as well as values for the kinetic constants, are presented.
Although there are many applications of fluorescamine to the assay of amino acids, peptides, proteins, and other primary amines, to obtain optimal utilization of this reagent, many details concerning the reaction still require investigation. The preceding paper (1) reports on the properties of fluorescamine and some amine derivatives, as well as on the influence of solvents, pH, and other factors in the formation of the fluorophors. The present report deals with studies on the kinetics of the fluorogenic reaction and of the hydrolysis of fluorescamine. MATERIALS
AND
METHODS
All amino acids and peptides were from SchwartzMann, Orangeburg, NY. Bovine serum albumin was from Pentex, Kankakee, IL. Spectra-grade acetone and histological-grade dioxane were from Fisher, Springfield, NJ. Fluorescamine (Fluram) was obtained from Hoffmann-La Roche, Inc., Nutley, NJ. All other chemicals were reagent grade from Fisher. Double-distilled water was used. Measurement of rates of reaction of fluorescamine with primary amines. An Aminco-Morrow stoppedflow fluorometer (American Instrument Co., Silver Spring, MD) was used to follow the reaction of primary amines with fluorescamine. The monochromator was set at 390 nm for excitation and a Wratten No. 65A secondary filter was used. The progress of the reaction was visualized on an oscilloscope, and a permanent record was made with a Polaroid camera. The scope of the experiments was
limited, because the stopped-flow instrument was equipped with two syringes that delivered equal volumes. Thus, the procedure in each experiment was to add fluorescamine in an organic solvent to an equal volume of buffered amine. The entire course of the reaction was followed with a slow scanning speed and the early phase of the reaction was followed with a fast scanning speed (Fig. 1). Maximal fluorescence was determined from the slow sweep and the time to reach half-maximal fluorescence (t,J was obtained from the fast sweep. Unless otherwise stated, all experiments were performed at 30°C. Except for the experiment in which the reagent concentration was varied, fluorescamine was prepared in the organic solvent at a concentration of I5 mg/IOO ml (0.5 mM). Measurement of hydrolysis rates. Fluorescamine is hydrolyzed in water to a nonfluorescent, nonfluorogenie product(s), which has not yet been characterized. Hydrolysis was followed by adding a solution of fluorescamine in an organic solvent to an aqueous buffer. After various intervals of time the remaining unhydrolyzed reagent was assayed by determining the fluorescence obtained when treated with an excess of peptide. While a 13 x loo-mm tube containing 0.8 ml of buffer was shaken vigorously on a vortex-type mixer, 1.0 ml of fluorescamine in an organic solvent was added. Shaking was continued and after a specific time interval, 0.20 ml of leucylalanine in water was added. Both reagents were introduced rapidly by means of glass syringes. A I-fold excess of leucylalanine (1.0 rmole) over the initial quantity of fluorescamine (0.25 pmole) was used for determining residual reagent. Measurements were made in an AmincoBowman spectrophotofluorometer at 390 nm excitation and 475 nm emission at room temperature. Un-
KINETICS
OF REACTION
AND HYDROLYSIS
less otherwise stated, the buffers were 0.2 M boric acid titrated to various pH values with sodium hydroxide. RESULTS
AND
DISCUSSION
Kinetics of reaction of fluorescamine with primary amines. Upon mixing an amine with fluorescamine in the stoppedflow fluorometer, fluorescence increased smoothly for several seconds to a maximal level (Fig. 1). Repeated tracings obtained with a given amine under the same conditions were essentially identical. When the data obtained from Fig. 1 were replotted as log (fnl - ft) vs time, where fm is the maximal fluorescence and ft is the fluorescence at a given time, a linear relationship was observed. This is demonstrated in Fig. 2 for various concentrations of fluorescamine. A rate constant (Iz,) can be obtained either as the slope of each logarithmic plot or can be more simply calculated as 0.691 t,. Since fluorescamine was in great excess over amine (initial concentrations of 0.5 mM and 20 PM, respectively in Fig. l), this linearity indicated first-order kinetics with respect to amine. As expected, k,, remained unchanged when the initial amine concentration was varied (lo-80 pM) and the fluorescamine concentration was kept constant, thereby corroborating the first-order kinetics with respect to amine.
FIG. 1. Reaction of alanine with fluorescamine in the stopped-flow fluorometer. In this illustration a 20-pM solution of alanine in .20 M sodium borate, pH 9.0, was mixed with an equal volume of fluorescamine in acetone (500 fiM) at 40°C. For the slow scan (upper tracing) and the fast scan (lower tracing) each major division on the absissa is equivalent to 5 set and 0.5 set, respectively. The ordinate represents relative fluorescence.
401
OF FLUORESCAMINE
The values of k, as determined from the slopes in Fig. 2 are neither constant nor proportional to the fluorescamine concentration. Indeed, k, appears to approach a limiting or saturation value. This behavior is suggestive of the following mechanism: F + ,-+,,.A,
-kA
I’,
1
where F is fluorescamine, A is the amine, [F.A] is an intermediate complex, P is the fluorescent product, and k ,, k 1, and k, are the appropriate rate constants. Assuming that there is a rapid equilibrium between the reactants and the intermediate complex as compared to the rate of product formation, that fluorescamine is in great excess and that the intermediate complex is nonfluorescent, it can be shown that: -dA, c@ k&z (F)(A,) -= dt k-,+k,(F) = --dt’ where AT is the sum of the amine concentrations present in free form and in the intermediate complex. Thus, the slope of each line in Fig. 2 is
hk, @‘)1/V-, + k, (FYI, or
FIG. 2. Time
course of the reaction
of fluorescaof flueoscilloscope tracings similar 1 were replotted as maximal fluorescence at a given time pM) was mixed with an equal in acetone at 30°C in the
mine with alanine at various concentrations rescamine: Data from to that depicted in Fig. fluorescence (fm) minus (fi) vs time. Alanine (20 volume of fluorescamine stopped-flow fluorometer.
402
STEIN,
BGHLEN,
AND UDENFRIEND
of several representative amines varied considerably (Fig. 4). Additionally k, del/k, = l/k, + k-,l[k,k, (F)] . creased for all amines with decreasing pH From the double-reciprocal plot based on (Fig. 4). The reaction rate was also found to this relationship (Fig. 3), k, is calculated as be dependent on the organic cosolvent 1 x 10’ set-’ and k,/k- 1 (equilibrium con- (Table I). Variation of temperature prostant) as 0.2 mM- ‘. These kinetic find- duced an apparent doubling in rate from ings are consistent with the following reac- 20°C to 50°C. The reaction rate was also affected by buffer salt. At pH 8.0 and 3O”C, k, was four times greater in 0.10 M sodium phosphate buffer than in 0.10 M sodium borate buffer. Fluorescamine, dissolved in methanol, reacts unusually slowly with primary amines, with half-times, at pH 9, of fractions of an hour rather than fractions of a second. This reduction in reaction rate may be explained by the report that alcoholic solvents form reversible addition products which can be rearranged
to
The rapid, reversible addition of a primary amine across the double bond of fluorescamine yields an intermediate that subsequently rearranges in a multistep sequence to the final fluorophor. There is a concomitant hydrolysis of reagent which limits the accuracy of the kinetic constants determined above. Influence of environment on rate of reaction. At a given pH, the reaction rates
PH
FIG. 4. Dependence of k, on pH with several representative amines: A solution of fluorescamine in acetone (2 mM) was mixed with an equal volume of buffered amine (20 +I) at 30°C. Bovine serum albumin was prepared at a concentration of 20 mg/lOO ml. (m) (Gly),, (0) Ala, (0) Arg, (A) Leu-Ala, (V) Asp, (0) BSA. TABLE
I
INFLUENCE OF ORGANIC COSOLVENT ON THE RATE OF REACTION OF FLUORESCAMINE WITH ALANINP
Solvent Acetone Acetonitrile Diglyme Dioxane FIG. 3. Double-reciprocal plot of fluorescamine concentration (F) and the observed pseudo-first-order rate constant (k,) determined at various concentrations of fluorescamine.
k, (set)-’ 1.6 0.6 1.6 2.3
a Equal volumes of fluorescamine (0.5 pM) in the appropriate solvent and alanine (20 jiM) in 0.20 M sodium borate, pH 9.0, were mixed at 30°C in the stopped-flow fluorometer.
KINETICS
OF REACTION
AND HYDROLYSIS
with fluorescamine, which are less reactive toward primary amines (1). Kinetics of hydrolysis of fluorescamine. Hydrolysis of fluorescamine was found to be first order with respect to fluorescamine, as evidenced by the linearity of logarithm fluorescence vs time (Fig. 5). The time for decrease of fluorescamine concentration to one half its initial value (t J was used to calculate the hydrolysis rate constant (k,,). The rate of hydrolysis of fluorescamine increased with increasing pH, irrespective of the organic cosolvent (Table II). At a given pH, however, k, depended on the particular cosolvent used in the reaction (Table II). Changing the acetone content from 10% to 50% produced a 4-fold decrease in k, at pH 9.0. Hydrolysis proceeded at less than half the rate in sodium phosphate compared to sodium borate buffer at pH 8.0, while a reduction in borate concentration from 0.10 M to 0.05 M at pH 9 resulted in a &fold increase in the rate of fluorescamine inactivation. Kinetic data are useful in understanding the reaction of fluorescamine with different primary amines. Qualitative correlations between reaction rates and extent of fluoro-
403
OF FLUORESCAMINE TABLE
II
OBSERVED RATE CONSTAXTS FOR HYDROLYSIS OF FLUORESCAMINE AS A FUNCTION OF pH AND ORGANIC COSOLVENT
pH/Solvent
7 8 9 10
k, (set ‘1 Acetone
Dioxane
Diglyme
Acetonitrile
0.008 0.016 0.073 0.210
0.58 I’ * ”
0.026 0.120 0.707 ”
0.003 0.007 0.013 0.064
ClThe rates were too rapid to permit measurement.
phor formation can be made. At pH 7.0, for example, aspartate yields little fluorescence, while glycine pentapeptide produces intense fluorescence (1). From Fig. 4 it is apparent that, at pH 7.0, the reaction rate for aspartate is low, whereas the reaction rate for glycine pentapeptide is high. Thus, at pH 7, aspartate, in contrast to glycine pentapeptide, does not compete well with the reaction in which fluorescamine is hydrolyzed to nonreactive products. The extent of fluorophor formation is not the only factor that influences the fluorescence attained with a given amine. The quantum yields of different fluorescamine derivatives measured under identical conditions vary greatly (1). The reaction environment not only affects the fluorophor formation, but also influences the fluorescence intensity of the fluorescamine derivatives, once formed (1). Differences in fluorescence attained in assaying various amines can be accounted for by a combination of the above factors. ACKNOWLEDGMENT
0L ---
5
..^~~~. IO TIME
- \
15
~J 20
(se<)
FIG. 5. Hydrolysis of fluorescamine: 1.0 ml of fluorescamine in diglyme (0.25 mM) was added to 0.8 ml of 0.20 M sodium borate, pH 8.0 (lower line), and at each time point the remaining reagent was titrated with 0.2 ml of leucylalanine (5 mM). Residual fluorescamine concentration is plotted as the fluorescence formed in arbitrary units. The upper line represents a similar experiment in which fluorescamine in acetone is hydrolyzed at pH 9.0.
The authors thank Dr. P. A. St. John of the American Instrument Company for providing and assisting with the operation of the stopped-flow apparatus. Thanks are also due to Drs. M. Nishikimi and D. Miller for aiding in the interpretation of the kinetic data and to Dr. M. Weigele for valuable discussions during the preparation of this manuscript. REFERENCE 1. DE
BERNARDO, S., WEIGELE, MANHART, K., LEIMGRUBER, STEIN, S., AND UDENFRIEND,
Biochen.
Biophys.
163,390-399.
M., W., S.
TOOME, V., BUHLEN, P.. (1974) Arch.
Studies
OF BIOCHEMISTRY
AND
BIOPHYSICS
on the Kinetics STANLEY
STEIN, Roche Institute
163, 400-403 (1974)
of Reaction PETER
and Hydrolysis
BiZHLEN,
of Molecular
Biology,
Received January
AND
SIDNEY
Nutley,
of Fluorescamine UDENFRIEND
New Jersey 07110
28, 1974
The influence of various parameters on the rate of reaction of fluorescamine with primary amines and on the rate of hydrolysis of the reagent is described. The studies indicate that both are dependent on the reaction conditions, includingpH, solvent in which the reagent is prepared, temperature, reagent concentration, and buffer salt. Under any set of conditions the reaction rates vary with the amines. A correlation between reaction rate and extent of fluorophor formation has been demonstrated. Kinetic evidence for a multistep reaction mechanism, as well as values for the kinetic constants, are presented.
Although there are many applications of fluorescamine to the assay of amino acids, peptides, proteins, and other primary amines, to obtain optimal utilization of this reagent, many details concerning the reaction still require investigation. The preceding paper (1) reports on the properties of fluorescamine and some amine derivatives, as well as on the influence of solvents, pH, and other factors in the formation of the fluorophors. The present report deals with studies on the kinetics of the fluorogenic reaction and of the hydrolysis of fluorescamine. MATERIALS
AND
METHODS
All amino acids and peptides were from SchwartzMann, Orangeburg, NY. Bovine serum albumin was from Pentex, Kankakee, IL. Spectra-grade acetone and histological-grade dioxane were from Fisher, Springfield, NJ. Fluorescamine (Fluram) was obtained from Hoffmann-La Roche, Inc., Nutley, NJ. All other chemicals were reagent grade from Fisher. Double-distilled water was used. Measurement of rates of reaction of fluorescamine with primary amines. An Aminco-Morrow stoppedflow fluorometer (American Instrument Co., Silver Spring, MD) was used to follow the reaction of primary amines with fluorescamine. The monochromator was set at 390 nm for excitation and a Wratten No. 65A secondary filter was used. The progress of the reaction was visualized on an oscilloscope, and a permanent record was made with a Polaroid camera. The scope of the experiments was
limited, because the stopped-flow instrument was equipped with two syringes that delivered equal volumes. Thus, the procedure in each experiment was to add fluorescamine in an organic solvent to an equal volume of buffered amine. The entire course of the reaction was followed with a slow scanning speed and the early phase of the reaction was followed with a fast scanning speed (Fig. 1). Maximal fluorescence was determined from the slow sweep and the time to reach half-maximal fluorescence (t,J was obtained from the fast sweep. Unless otherwise stated, all experiments were performed at 30°C. Except for the experiment in which the reagent concentration was varied, fluorescamine was prepared in the organic solvent at a concentration of I5 mg/IOO ml (0.5 mM). Measurement of hydrolysis rates. Fluorescamine is hydrolyzed in water to a nonfluorescent, nonfluorogenie product(s), which has not yet been characterized. Hydrolysis was followed by adding a solution of fluorescamine in an organic solvent to an aqueous buffer. After various intervals of time the remaining unhydrolyzed reagent was assayed by determining the fluorescence obtained when treated with an excess of peptide. While a 13 x loo-mm tube containing 0.8 ml of buffer was shaken vigorously on a vortex-type mixer, 1.0 ml of fluorescamine in an organic solvent was added. Shaking was continued and after a specific time interval, 0.20 ml of leucylalanine in water was added. Both reagents were introduced rapidly by means of glass syringes. A I-fold excess of leucylalanine (1.0 rmole) over the initial quantity of fluorescamine (0.25 pmole) was used for determining residual reagent. Measurements were made in an AmincoBowman spectrophotofluorometer at 390 nm excitation and 475 nm emission at room temperature. Un-
KINETICS
OF REACTION
AND HYDROLYSIS
less otherwise stated, the buffers were 0.2 M boric acid titrated to various pH values with sodium hydroxide. RESULTS
AND
DISCUSSION
Kinetics of reaction of fluorescamine with primary amines. Upon mixing an amine with fluorescamine in the stoppedflow fluorometer, fluorescence increased smoothly for several seconds to a maximal level (Fig. 1). Repeated tracings obtained with a given amine under the same conditions were essentially identical. When the data obtained from Fig. 1 were replotted as log (fnl - ft) vs time, where fm is the maximal fluorescence and ft is the fluorescence at a given time, a linear relationship was observed. This is demonstrated in Fig. 2 for various concentrations of fluorescamine. A rate constant (Iz,) can be obtained either as the slope of each logarithmic plot or can be more simply calculated as 0.691 t,. Since fluorescamine was in great excess over amine (initial concentrations of 0.5 mM and 20 PM, respectively in Fig. l), this linearity indicated first-order kinetics with respect to amine. As expected, k,, remained unchanged when the initial amine concentration was varied (lo-80 pM) and the fluorescamine concentration was kept constant, thereby corroborating the first-order kinetics with respect to amine.
FIG. 1. Reaction of alanine with fluorescamine in the stopped-flow fluorometer. In this illustration a 20-pM solution of alanine in .20 M sodium borate, pH 9.0, was mixed with an equal volume of fluorescamine in acetone (500 fiM) at 40°C. For the slow scan (upper tracing) and the fast scan (lower tracing) each major division on the absissa is equivalent to 5 set and 0.5 set, respectively. The ordinate represents relative fluorescence.
401
OF FLUORESCAMINE
The values of k, as determined from the slopes in Fig. 2 are neither constant nor proportional to the fluorescamine concentration. Indeed, k, appears to approach a limiting or saturation value. This behavior is suggestive of the following mechanism: F + ,-+,,.A,
-kA
I’,
1
where F is fluorescamine, A is the amine, [F.A] is an intermediate complex, P is the fluorescent product, and k ,, k 1, and k, are the appropriate rate constants. Assuming that there is a rapid equilibrium between the reactants and the intermediate complex as compared to the rate of product formation, that fluorescamine is in great excess and that the intermediate complex is nonfluorescent, it can be shown that: -dA, c@ k&z (F)(A,) -= dt k-,+k,(F) = --dt’ where AT is the sum of the amine concentrations present in free form and in the intermediate complex. Thus, the slope of each line in Fig. 2 is
hk, @‘)1/V-, + k, (FYI, or
FIG. 2. Time
course of the reaction
of fluorescaof flueoscilloscope tracings similar 1 were replotted as maximal fluorescence at a given time pM) was mixed with an equal in acetone at 30°C in the
mine with alanine at various concentrations rescamine: Data from to that depicted in Fig. fluorescence (fm) minus (fi) vs time. Alanine (20 volume of fluorescamine stopped-flow fluorometer.
402
STEIN,
BGHLEN,
AND UDENFRIEND
of several representative amines varied considerably (Fig. 4). Additionally k, del/k, = l/k, + k-,l[k,k, (F)] . creased for all amines with decreasing pH From the double-reciprocal plot based on (Fig. 4). The reaction rate was also found to this relationship (Fig. 3), k, is calculated as be dependent on the organic cosolvent 1 x 10’ set-’ and k,/k- 1 (equilibrium con- (Table I). Variation of temperature prostant) as 0.2 mM- ‘. These kinetic find- duced an apparent doubling in rate from ings are consistent with the following reac- 20°C to 50°C. The reaction rate was also affected by buffer salt. At pH 8.0 and 3O”C, k, was four times greater in 0.10 M sodium phosphate buffer than in 0.10 M sodium borate buffer. Fluorescamine, dissolved in methanol, reacts unusually slowly with primary amines, with half-times, at pH 9, of fractions of an hour rather than fractions of a second. This reduction in reaction rate may be explained by the report that alcoholic solvents form reversible addition products which can be rearranged
to
The rapid, reversible addition of a primary amine across the double bond of fluorescamine yields an intermediate that subsequently rearranges in a multistep sequence to the final fluorophor. There is a concomitant hydrolysis of reagent which limits the accuracy of the kinetic constants determined above. Influence of environment on rate of reaction. At a given pH, the reaction rates
PH
FIG. 4. Dependence of k, on pH with several representative amines: A solution of fluorescamine in acetone (2 mM) was mixed with an equal volume of buffered amine (20 +I) at 30°C. Bovine serum albumin was prepared at a concentration of 20 mg/lOO ml. (m) (Gly),, (0) Ala, (0) Arg, (A) Leu-Ala, (V) Asp, (0) BSA. TABLE
I
INFLUENCE OF ORGANIC COSOLVENT ON THE RATE OF REACTION OF FLUORESCAMINE WITH ALANINP
Solvent Acetone Acetonitrile Diglyme Dioxane FIG. 3. Double-reciprocal plot of fluorescamine concentration (F) and the observed pseudo-first-order rate constant (k,) determined at various concentrations of fluorescamine.
k, (set)-’ 1.6 0.6 1.6 2.3
a Equal volumes of fluorescamine (0.5 pM) in the appropriate solvent and alanine (20 jiM) in 0.20 M sodium borate, pH 9.0, were mixed at 30°C in the stopped-flow fluorometer.
KINETICS
OF REACTION
AND HYDROLYSIS
with fluorescamine, which are less reactive toward primary amines (1). Kinetics of hydrolysis of fluorescamine. Hydrolysis of fluorescamine was found to be first order with respect to fluorescamine, as evidenced by the linearity of logarithm fluorescence vs time (Fig. 5). The time for decrease of fluorescamine concentration to one half its initial value (t J was used to calculate the hydrolysis rate constant (k,,). The rate of hydrolysis of fluorescamine increased with increasing pH, irrespective of the organic cosolvent (Table II). At a given pH, however, k, depended on the particular cosolvent used in the reaction (Table II). Changing the acetone content from 10% to 50% produced a 4-fold decrease in k, at pH 9.0. Hydrolysis proceeded at less than half the rate in sodium phosphate compared to sodium borate buffer at pH 8.0, while a reduction in borate concentration from 0.10 M to 0.05 M at pH 9 resulted in a &fold increase in the rate of fluorescamine inactivation. Kinetic data are useful in understanding the reaction of fluorescamine with different primary amines. Qualitative correlations between reaction rates and extent of fluoro-
403
OF FLUORESCAMINE TABLE
II
OBSERVED RATE CONSTAXTS FOR HYDROLYSIS OF FLUORESCAMINE AS A FUNCTION OF pH AND ORGANIC COSOLVENT
pH/Solvent
7 8 9 10
k, (set ‘1 Acetone
Dioxane
Diglyme
Acetonitrile
0.008 0.016 0.073 0.210
0.58 I’ * ”
0.026 0.120 0.707 ”
0.003 0.007 0.013 0.064
ClThe rates were too rapid to permit measurement.
phor formation can be made. At pH 7.0, for example, aspartate yields little fluorescence, while glycine pentapeptide produces intense fluorescence (1). From Fig. 4 it is apparent that, at pH 7.0, the reaction rate for aspartate is low, whereas the reaction rate for glycine pentapeptide is high. Thus, at pH 7, aspartate, in contrast to glycine pentapeptide, does not compete well with the reaction in which fluorescamine is hydrolyzed to nonreactive products. The extent of fluorophor formation is not the only factor that influences the fluorescence attained with a given amine. The quantum yields of different fluorescamine derivatives measured under identical conditions vary greatly (1). The reaction environment not only affects the fluorophor formation, but also influences the fluorescence intensity of the fluorescamine derivatives, once formed (1). Differences in fluorescence attained in assaying various amines can be accounted for by a combination of the above factors. ACKNOWLEDGMENT
0L ---
5
..^~~~. IO TIME
- \
15
~J 20
(se<)
FIG. 5. Hydrolysis of fluorescamine: 1.0 ml of fluorescamine in diglyme (0.25 mM) was added to 0.8 ml of 0.20 M sodium borate, pH 8.0 (lower line), and at each time point the remaining reagent was titrated with 0.2 ml of leucylalanine (5 mM). Residual fluorescamine concentration is plotted as the fluorescence formed in arbitrary units. The upper line represents a similar experiment in which fluorescamine in acetone is hydrolyzed at pH 9.0.
The authors thank Dr. P. A. St. John of the American Instrument Company for providing and assisting with the operation of the stopped-flow apparatus. Thanks are also due to Drs. M. Nishikimi and D. Miller for aiding in the interpretation of the kinetic data and to Dr. M. Weigele for valuable discussions during the preparation of this manuscript. REFERENCE 1. DE
BERNARDO, S., WEIGELE, MANHART, K., LEIMGRUBER, STEIN, S., AND UDENFRIEND,
Biochen.
Biophys.
163,390-399.
M., W., S.
TOOME, V., BUHLEN, P.. (1974) Arch.