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A direct colorimetric method for the study of myosin ATPase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS ~03, 111-118
(1964)
A Direct Colorimetric Method for the Study of Myosin ATPase ~ PATRICIA RAINFORD From the Cardiovascular Research Institute, University of California, San Francisco, California ~ Received J u l y 18, 1963 At 20~ in 0.6 M KCI, the largest difference between the acidic and basic optical densities of t h e dye, p-phenylazo-phenol sulfonie acid, occurs at X = 425 mt~, and t h e dye has a p K of 8. Around p H 8, A T P hydrolysis yields 1 mole H+/1 mole A T P . Therefore, in this pH range, it is possible to follow r a t e of p r o t o n production, i.e., ATPase activity, b y following O.D.42~ as a function of time. R a t e s thus measured are linear functions of rates measured b y the conventional o r t h o p h o s p h a t e m e t h o d used for calibration, a n d the s e n s i t i v i t y of t h e O.D. m e t h o d is comparable to t h a t of t h e c o n v e n t i o n a l method. A p a r t from its convenience in routine work the m e t h o d lends itself to mass applications as in collecting fractions, or to t r a n s i e n t m e a s u r e m e n t s as originally devised b y G u t f r e u n d and S t u r t e v a n t for c h y m o t r y p s i n . INTRODUCTION
Our laboratory has frequently been concerned with kinetic studies of the myosin catalyzed dephosphorylation of adenosine triphosphate (ATP). In the neighborhood of pH 8, which is our standard condition, the reaction may be written as: A T P ~- + H~O --* A D P 3- + P~- + H +
There are several well-established methods for studying this reaction, e.g., ehemieal assay for orthophosphate (1, 2); alkaline neutralization of the hydrogen ion produced using a pH star; interferometry (3); etc. However, if the number of samples to be analyzed is very large, as in chromatographic purification of enzyme, or if the problem is fast kinetics, then these existing methods all Mve serious technical disadvantages. Colorimetry, as first shown for chynlotrypsin (4), offers a way around these difficulties. A dye whose pK is very nearly 1 This p a p e r was p r e s e n t e d orally b y P a t r i c i a Rainford at the meeting of t h e F e d e r a t i o n of American Societies for E x p e r i m e n t a l Biology, A t l a n t i c City, New Jersey, April 17, 1963. 2 This research was s u p p o r t e d by grants from t h e N a t i o n a l Science F o u n d a t i o n and t h e American H e a r t Association.
the pH at which the reaction is to be studied will have the property of suffering large changes in absorbance for very small changes in pH. If the concern is with small amounts of reaction, if the activity is not too sensitive to pH at the pH in question, and if the dye does not affect the enzyme, then such a dye system provides a rapid and convenient means for studying the kinetics of ATP hydrolysis. The dye p-phenylazo-phenol sulfonic acid, used in the form of the sodium salt, meets all the necessary requirements for myosin catalysis at pH 8. 3 MATERIALS AND METHODS Myosin A was p r e p a r e d from r a b b i t back muscle by t h e s t a n d a r d m e t h o d in this l a b o r a t o r y . F o r all experimental purposes a final c o n c e n t r a t i o n of a b o u t 0.001% in 0.6 M KC1 was used. 3 J u s t before the p r e s e n t a t i o n of this paper, Dr. Wilfried M o m m a e r t s kindly called to our att e n t i o n a paper, "Screening of chicks with e r y t h romyeloblastic leukosis for p l a s m a a c t i v i t y in d e p h o s p h o r y l a t i o n , " in which the authors investigated the A T P a s e a c t i v i t y of virus particles b y the discoloration of b r o m 4 h y m o l blue, which has a p K of 7.0. While the objectives of these authors were r a t h e r different from ours, t h e i r work m i g h t well h a v e been a d a p t e d to our problem. 111
112
RAINFORD may damage the enzyme and lead to inconsistent results. It was found advantageous to keep the volume of protein added to the dye solution always the same. This avoids any correction factor for dilution. In cases where the myosin was too concentrated, the sample was diluted with 0.6 M KC1, and the experiment repeated using the same volumes as before. Crystalline Na~H~ATP was obtained from Sigma Co., and PCMB-Na salt from Calbiochem; other chemicals used were reagent grade from J. T. Baker Co. In the last experiment described, the analogue computer used was an Electronic Associates TR.48, the log converter was from Houston Instruments Corp., and the recorder from Photovolt Corp.
Stock dye solutions were prepared i n 0.6 M KC1 containing 1 mM ATP and 10 mM Ca ~+ and brought to pH 9. The pH of this solution was checked at regular intervals as the solution is unbuffered and the pH tends to drift. The dye, which is readily soluble in alkaline solution, was made up daily, and each preparation was filtered to remove any undissolved solid. Each time a fresh stock dye solution was prepared it was necessary to plot a control of optical density versus time in seconds, after addition of ATP. This established the linear range for a specific dye solution, and once obtained, it was then only necessary to record the time taken for a specific O.D. change, within that range, for each myosin aliquot. All O.D. measurements were made with a Zeiss P.M.Q. II spectrophotometer, using 1 cm. cells containing 2.5 ml. of the dye solution and a 0.5-ml. aliquot of the myosin, treated or untreated. The volumes used may be adjusted to suit the experiment, but for our purposes the above were most convenient. Protein solutions were kept at pH 7 and added to the reaction mixture at pH 9. The pH was never adjusted when protein was present, as this
RESULTS AND DISCUSSION T h e visible sp ect r a of t h e acidic a n d basic f o r m s of t h e d y e (Fig. 1) show clearly t h a t a large c h a n g e in a b s o r b a n c e w i t h i o n i z a t i o n can be e x p e c t e d if t h e O,D. of t h e d y e s o l u t i o n is m e a s u r e d a t 425 m~. T h e p K
0.25
NQS 0 3 " ~ - N =N - - O -
OH
~ - - P H E N Y L A Z O --PHENOL.
~pH S.O
0~0
47.5 im~ 0.1S O.D.
0.10
0.05
\/ ~e
300
~00
i
!
i
500
600
700
Fro. 1. Spectra of the acidic and basic forms of p-phenylazo-phenol sulfonic acid; in 0.6 M KCI ;Em at 425 m~; pH 9 = 1.73 X 104.
MYOSIN ATPASE of the dye at 20~ is 8 (Fig. 2), and the change of optical density with p H is linear in the region of p H 8.6-7.6. For the measurement of ATPase rates,
113
stock dye solutions in 0.6 M KC1 were prepared that contained metaI ions and A T P in the required amounts. If the p H of these solutions was poised at 9, and protein
t.o-
0~ D~
1.5-
I
J 0
2
,4
6
8
10
12
14
pH FiG. 2. Speetrophotometric titration at 425 m~; 20~ sulfonic acid in 100 ml. of 0.6 M KC1.
1.3 rag. of p-phenylazo-phenol
0.3
QO.
0.2
0.1
I
0
100
I
200 SECONDS
9
300
l
400
I
500
F~G. 3. O.D.425m# as a function of time for different myosin concentrations. The enzyme concentrations shown are in the ratio 1:2:4. This is also the ratio of the slopes (ATPase rates) of the curves.
114
RAINFORD
at neutral pH added, then the pH of the mixture fell, either rapidly or more slowly depending on the protein concentration, into the linear O.D. vs. time range for the system. The change in O.D. was then followed with time and plotted. A reaction which was begun with the system poised in the way mentioned, under conditions in which the reaction kinetics were expected to be of zero order, demonstrated that O.D. was indeed a linear function of time (Fig. 3). These data also showed that the reaction rate, measured by the dye method, was strictly proportional to the enzyme concentration, By using different enzyme concentrations, different ATPase rates were obtained. These rates were measured by the dye method and by our standard orthophosphate method. The results were plotted in the form of a
calibration curve (Fig. 4), which may be considered to be a validation of the method, and which shows that with the convenient dye concentration used here, 2.6 O.D. units by the orthophosphate assay correspond to 1 O.D. unit by the dye method. It is obvious that the more concentrated the dye solution, the greater the O.D. change in the linear range of the dye. If a calibration curve is to be of use at all times for the calculation of absolute amounts of inorganic phosphate split, then some correction must be employed for different dye solutions. There are two ways to do this: Either all dye solutions prepared must be the same concentration (this would prove tedious and time consuming, as only micro amounts of the dye are used); or, if the O.D. of each dye solution prepared is known at one pH (say 9), then readings from the
0.4
0.3 :2 m l
Pl
MYO3/N
A
0.2
A
0.1 0.5" rnl
MYO3/N
S'
0.05
( A'r
A
I
I
0.10
O. IS
oo
/Dye
FIG. 4. Validation and calibration of the dye m e t h o d with t h e s t a n d a r d o r t h o p h o s p h a t e assay. On t h e figure are indicated t h e n u m b e r of milliliters of stock myosin solution added to 20 ml, of solvent.
0.5
0.4.
I.L "t SEC
0.3
REACTION MEASURED BETWEEN 0.0.: .45 O.D.= .40 0.2
200
100
D I LUTI
tOO 0NS
200
FI~. 5. '/'Mock elution curve" obtained from a series of aliquots of different myosin concentration. ( C ) - - 9 Taken from complete plots of O.D. versus time for a specific AO.D.~5 ; ( e - - - - e ) A simplification of the above, obtained from just two absorbance readings in the spectrophotometer, i.e, the same/~O.D.~.
0.6
0.,5 (11).
~IIMB
TREATMENT)
0.4
I 0
"100
li"
!
(
200 300 S ~"CONDS
I
400
500
]?~G. 6. PCMB-induced transients, as followed with the dye method. Myosin at 0.38 rag. per milliliter was pretreated with 6-7 moles PCMB/105 g. The lower limit of the linear range for this dye solution is indicated by the control plot. 115
116
RAINFORD
calibration curve can be corrected by the O.D. ratio. This second alternative is relatively simple. The over-all result is that the absolute concentration of any dye solution prepared, in terms of moles per liter, need not be known, and the strength of a dye solution suitable for use can merely be judged by eye. In a separate series of experiments, using a p H star, it was shown that the dye, at all concentrations used here, had no effect on the myosin ATPase activity. The foregoing method has several useful applications. The first of these is in assaying for ATPase activity when myosin is being
purified by chromatography, i.e., when a large number of protein aliquots must be analyzed. A sufficient amount of stock dye solution was prepared and the linear range established from a control plot of 0.D.425 vs. time in seconds. Once this was known it was only necessary to record the time taken for a specific O.D. change, within the linear range, for each myosin aliquot. Figure 5 shows the distribution of ATPase activity in a " m o c k " elution curve, i.e., from a series of tubes of varying myosin concentration. The readings could have been made with a simple colorimeter, because, for the scanning of an
0.50 Using IOk of 40p. M ATP in IOml "dye" sol. end then 2.5 ml. of this in 3.0 ml. [Protein] = i_~._~. % 1 X . _~4_0.5 .~.O6
E
"
~= 0 . 3 0
~
I i~_q50_ ~
~ ~,
"%,j
-'......
Q.
o
Limit of linearity
0.20 --
1 I
I
0 I
I
5 Time
I
9 I0 in minutes
I
15
I
I
19 20
I
__
23
Fro. 7. Aa actual recording of a slow transient in ATPase activity resulting from desaturation by substrate. 10 X of 40 mM ATP were mixed with 10 ml. of dye solution. 2.5 ml. of this were treated with 0.5 rot. of myosin. Myosin concentration was 0.0024%.
MYOSIN ATPASE elution curve, the method in its simplest form is entirely adequate. The method was also applied to following slow transients in ATPase activity. For example, Gilmour and Gellert (5) showed that the decrease in activity with time, after myosin had been treated with P C M B , was accelerated in the presence of substrate. The series of curves obtained (Fig. 6) shows clearly the initial acceleration of ATPase activity and also the decay of activity with
117
increased time of incubation. The "curving off" which occurred before the limit of the linear range had been reached indicates quite adequately the acceleration of the decrease in activity occurring in the presence of ATP. A further application was to the continuous monitoring of a slow transient in ATPase activity. The transient followed was the total dephosphorylation of a small quantity of A T P by a comparatively large quantity of myosin (Fig. 7). For this
Phosphate production with limiting ATP (From 'dye' recording)
0.5
----~
0.2
....... ~.................................. f ' ........................
/
"~
J . 5p3hXslpOh-::p~erS i it e r
o
I
0
l
. . . . .
I.
I
15
30 45 60 Time in minufes FI6.8. Replotting of Fig. 7, together with a comparable curve obtained with the orthophosphate method. ( - - ) The conversion from the dye recording of Fig. 7; (..... ) orthophosphate measurement. Arrows mark the region analyzed by the analogue computer.
OF" P R O D U C T
FORMATION
VS.
TIME
>. t~j . 2 4 1
f
.229
q
.217 .205
I-..193 .181
I
I
I
2t.0 6.0 9.0 T I M E IN M I N U T I ~ S T I M E IN M I N U T E S Fro. 9. The fitting of the slow transient by an analogue computer working on repetitive operation. Left: the recorded transient; right: the oscilloscope trace. 0
118
RAINFORD
purpose the output of the spectrophotometer was fed into a log converter (to change per cent transmission into optical density). The output of the converter was amplified and recorded on a linear scale. The transient obtained followed closely the transient in the same system using the orthophosphate method (Fig. 8). T o illustrate a use of such recordings, the transient was fitted b y an analogue computer, working on repetitive operation, to the differential equation of the scheme: E+S. where
kl k-1
Km
"X
ks --~ E + P,
L I + k~ kl
T h e result obtained (Fig. 9) gave a value of 2.99 X 10-5 M for the constant Kw. This is a very reasonable result for the K~ of myosin, obtained from an experiment at a single substrate concentration. CONCLUSION We feel t h a t the foregoing method is convenient for routine A T P a s e activity determinations. Each run takes up to 5 minutes, and consequently a very large
number of samples can be dealt with in a n y one d a y b y one person. This is in contrast to the m a x i m u m of 18 runs, under the same conditions, using the orthophosphate method or the p H stat. This clear advantage does not count the simplicity in glassware, pipetting, etc. I n addition, by using a rapid mixing chamber, the dye method described should be readily adaptable to recording fast initial transients. ACKNOWLEDGMENTS The author wishes to express her gratitude to Professor Manuel Morales for useful discussion of this work. She is also indebted to Professor I. M. Klotz who kindly gave her the dye, and to Dr. Charles Walter for assistance in obtaining the analogue computer analysis. REFERENCES 1. FISK~, C. H., AND SUEBA Row, Y., J. Biol. Chem. 66, 375 (1925). 2. MARTIN,J. B., AND DOTY, D. M., Anal. Chem. 21,965 (1949). 3. ASAI, H., AND MORALES,M. F., Arch. Biochem. Biophys. 99, 383 (1962). 4. GUTFREUND, ~-L, AND STURTEVANT, J. M., Biochem. J. 63, 656 (1956). 5. GILMOVR,D., ANDGEL~ER%M., Arch. Biochem. Biophys. 93, 605 (1961).
(1964)
A Direct Colorimetric Method for the Study of Myosin ATPase ~ PATRICIA RAINFORD From the Cardiovascular Research Institute, University of California, San Francisco, California ~ Received J u l y 18, 1963 At 20~ in 0.6 M KCI, the largest difference between the acidic and basic optical densities of t h e dye, p-phenylazo-phenol sulfonie acid, occurs at X = 425 mt~, and t h e dye has a p K of 8. Around p H 8, A T P hydrolysis yields 1 mole H+/1 mole A T P . Therefore, in this pH range, it is possible to follow r a t e of p r o t o n production, i.e., ATPase activity, b y following O.D.42~ as a function of time. R a t e s thus measured are linear functions of rates measured b y the conventional o r t h o p h o s p h a t e m e t h o d used for calibration, a n d the s e n s i t i v i t y of t h e O.D. m e t h o d is comparable to t h a t of t h e c o n v e n t i o n a l method. A p a r t from its convenience in routine work the m e t h o d lends itself to mass applications as in collecting fractions, or to t r a n s i e n t m e a s u r e m e n t s as originally devised b y G u t f r e u n d and S t u r t e v a n t for c h y m o t r y p s i n . INTRODUCTION
Our laboratory has frequently been concerned with kinetic studies of the myosin catalyzed dephosphorylation of adenosine triphosphate (ATP). In the neighborhood of pH 8, which is our standard condition, the reaction may be written as: A T P ~- + H~O --* A D P 3- + P~- + H +
There are several well-established methods for studying this reaction, e.g., ehemieal assay for orthophosphate (1, 2); alkaline neutralization of the hydrogen ion produced using a pH star; interferometry (3); etc. However, if the number of samples to be analyzed is very large, as in chromatographic purification of enzyme, or if the problem is fast kinetics, then these existing methods all Mve serious technical disadvantages. Colorimetry, as first shown for chynlotrypsin (4), offers a way around these difficulties. A dye whose pK is very nearly 1 This p a p e r was p r e s e n t e d orally b y P a t r i c i a Rainford at the meeting of t h e F e d e r a t i o n of American Societies for E x p e r i m e n t a l Biology, A t l a n t i c City, New Jersey, April 17, 1963. 2 This research was s u p p o r t e d by grants from t h e N a t i o n a l Science F o u n d a t i o n and t h e American H e a r t Association.
the pH at which the reaction is to be studied will have the property of suffering large changes in absorbance for very small changes in pH. If the concern is with small amounts of reaction, if the activity is not too sensitive to pH at the pH in question, and if the dye does not affect the enzyme, then such a dye system provides a rapid and convenient means for studying the kinetics of ATP hydrolysis. The dye p-phenylazo-phenol sulfonic acid, used in the form of the sodium salt, meets all the necessary requirements for myosin catalysis at pH 8. 3 MATERIALS AND METHODS Myosin A was p r e p a r e d from r a b b i t back muscle by t h e s t a n d a r d m e t h o d in this l a b o r a t o r y . F o r all experimental purposes a final c o n c e n t r a t i o n of a b o u t 0.001% in 0.6 M KC1 was used. 3 J u s t before the p r e s e n t a t i o n of this paper, Dr. Wilfried M o m m a e r t s kindly called to our att e n t i o n a paper, "Screening of chicks with e r y t h romyeloblastic leukosis for p l a s m a a c t i v i t y in d e p h o s p h o r y l a t i o n , " in which the authors investigated the A T P a s e a c t i v i t y of virus particles b y the discoloration of b r o m 4 h y m o l blue, which has a p K of 7.0. While the objectives of these authors were r a t h e r different from ours, t h e i r work m i g h t well h a v e been a d a p t e d to our problem. 111
112
RAINFORD may damage the enzyme and lead to inconsistent results. It was found advantageous to keep the volume of protein added to the dye solution always the same. This avoids any correction factor for dilution. In cases where the myosin was too concentrated, the sample was diluted with 0.6 M KC1, and the experiment repeated using the same volumes as before. Crystalline Na~H~ATP was obtained from Sigma Co., and PCMB-Na salt from Calbiochem; other chemicals used were reagent grade from J. T. Baker Co. In the last experiment described, the analogue computer used was an Electronic Associates TR.48, the log converter was from Houston Instruments Corp., and the recorder from Photovolt Corp.
Stock dye solutions were prepared i n 0.6 M KC1 containing 1 mM ATP and 10 mM Ca ~+ and brought to pH 9. The pH of this solution was checked at regular intervals as the solution is unbuffered and the pH tends to drift. The dye, which is readily soluble in alkaline solution, was made up daily, and each preparation was filtered to remove any undissolved solid. Each time a fresh stock dye solution was prepared it was necessary to plot a control of optical density versus time in seconds, after addition of ATP. This established the linear range for a specific dye solution, and once obtained, it was then only necessary to record the time taken for a specific O.D. change, within that range, for each myosin aliquot. All O.D. measurements were made with a Zeiss P.M.Q. II spectrophotometer, using 1 cm. cells containing 2.5 ml. of the dye solution and a 0.5-ml. aliquot of the myosin, treated or untreated. The volumes used may be adjusted to suit the experiment, but for our purposes the above were most convenient. Protein solutions were kept at pH 7 and added to the reaction mixture at pH 9. The pH was never adjusted when protein was present, as this
RESULTS AND DISCUSSION T h e visible sp ect r a of t h e acidic a n d basic f o r m s of t h e d y e (Fig. 1) show clearly t h a t a large c h a n g e in a b s o r b a n c e w i t h i o n i z a t i o n can be e x p e c t e d if t h e O,D. of t h e d y e s o l u t i o n is m e a s u r e d a t 425 m~. T h e p K
0.25
NQS 0 3 " ~ - N =N - - O -
OH
~ - - P H E N Y L A Z O --PHENOL.
~pH S.O
0~0
47.5 im~ 0.1S O.D.
0.10
0.05
\/ ~e
300
~00
i
!
i
500
600
700
Fro. 1. Spectra of the acidic and basic forms of p-phenylazo-phenol sulfonic acid; in 0.6 M KCI ;Em at 425 m~; pH 9 = 1.73 X 104.
MYOSIN ATPASE of the dye at 20~ is 8 (Fig. 2), and the change of optical density with p H is linear in the region of p H 8.6-7.6. For the measurement of ATPase rates,
113
stock dye solutions in 0.6 M KC1 were prepared that contained metaI ions and A T P in the required amounts. If the p H of these solutions was poised at 9, and protein
t.o-
0~ D~
1.5-
I
J 0
2
,4
6
8
10
12
14
pH FiG. 2. Speetrophotometric titration at 425 m~; 20~ sulfonic acid in 100 ml. of 0.6 M KC1.
1.3 rag. of p-phenylazo-phenol
0.3
QO.
0.2
0.1
I
0
100
I
200 SECONDS
9
300
l
400
I
500
F~G. 3. O.D.425m# as a function of time for different myosin concentrations. The enzyme concentrations shown are in the ratio 1:2:4. This is also the ratio of the slopes (ATPase rates) of the curves.
114
RAINFORD
at neutral pH added, then the pH of the mixture fell, either rapidly or more slowly depending on the protein concentration, into the linear O.D. vs. time range for the system. The change in O.D. was then followed with time and plotted. A reaction which was begun with the system poised in the way mentioned, under conditions in which the reaction kinetics were expected to be of zero order, demonstrated that O.D. was indeed a linear function of time (Fig. 3). These data also showed that the reaction rate, measured by the dye method, was strictly proportional to the enzyme concentration, By using different enzyme concentrations, different ATPase rates were obtained. These rates were measured by the dye method and by our standard orthophosphate method. The results were plotted in the form of a
calibration curve (Fig. 4), which may be considered to be a validation of the method, and which shows that with the convenient dye concentration used here, 2.6 O.D. units by the orthophosphate assay correspond to 1 O.D. unit by the dye method. It is obvious that the more concentrated the dye solution, the greater the O.D. change in the linear range of the dye. If a calibration curve is to be of use at all times for the calculation of absolute amounts of inorganic phosphate split, then some correction must be employed for different dye solutions. There are two ways to do this: Either all dye solutions prepared must be the same concentration (this would prove tedious and time consuming, as only micro amounts of the dye are used); or, if the O.D. of each dye solution prepared is known at one pH (say 9), then readings from the
0.4
0.3 :2 m l
Pl
MYO3/N
A
0.2
A
0.1 0.5" rnl
MYO3/N
S'
0.05
( A'r
A
I
I
0.10
O. IS
oo
/Dye
FIG. 4. Validation and calibration of the dye m e t h o d with t h e s t a n d a r d o r t h o p h o s p h a t e assay. On t h e figure are indicated t h e n u m b e r of milliliters of stock myosin solution added to 20 ml, of solvent.
0.5
0.4.
I.L "t SEC
0.3
REACTION MEASURED BETWEEN 0.0.: .45 O.D.= .40 0.2
200
100
D I LUTI
tOO 0NS
200
FI~. 5. '/'Mock elution curve" obtained from a series of aliquots of different myosin concentration. ( C ) - - 9 Taken from complete plots of O.D. versus time for a specific AO.D.~5 ; ( e - - - - e ) A simplification of the above, obtained from just two absorbance readings in the spectrophotometer, i.e, the same/~O.D.~.
0.6
0.,5 (11).
~IIMB
TREATMENT)
0.4
I 0
"100
li"
!
(
200 300 S ~"CONDS
I
400
500
]?~G. 6. PCMB-induced transients, as followed with the dye method. Myosin at 0.38 rag. per milliliter was pretreated with 6-7 moles PCMB/105 g. The lower limit of the linear range for this dye solution is indicated by the control plot. 115
116
RAINFORD
calibration curve can be corrected by the O.D. ratio. This second alternative is relatively simple. The over-all result is that the absolute concentration of any dye solution prepared, in terms of moles per liter, need not be known, and the strength of a dye solution suitable for use can merely be judged by eye. In a separate series of experiments, using a p H star, it was shown that the dye, at all concentrations used here, had no effect on the myosin ATPase activity. The foregoing method has several useful applications. The first of these is in assaying for ATPase activity when myosin is being
purified by chromatography, i.e., when a large number of protein aliquots must be analyzed. A sufficient amount of stock dye solution was prepared and the linear range established from a control plot of 0.D.425 vs. time in seconds. Once this was known it was only necessary to record the time taken for a specific O.D. change, within the linear range, for each myosin aliquot. Figure 5 shows the distribution of ATPase activity in a " m o c k " elution curve, i.e., from a series of tubes of varying myosin concentration. The readings could have been made with a simple colorimeter, because, for the scanning of an
0.50 Using IOk of 40p. M ATP in IOml "dye" sol. end then 2.5 ml. of this in 3.0 ml. [Protein] = i_~._~. % 1 X . _~4_0.5 .~.O6
E
"
~= 0 . 3 0
~
I i~_q50_ ~
~ ~,
"%,j
-'......
Q.
o
Limit of linearity
0.20 --
1 I
I
0 I
I
5 Time
I
9 I0 in minutes
I
15
I
I
19 20
I
__
23
Fro. 7. Aa actual recording of a slow transient in ATPase activity resulting from desaturation by substrate. 10 X of 40 mM ATP were mixed with 10 ml. of dye solution. 2.5 ml. of this were treated with 0.5 rot. of myosin. Myosin concentration was 0.0024%.
MYOSIN ATPASE elution curve, the method in its simplest form is entirely adequate. The method was also applied to following slow transients in ATPase activity. For example, Gilmour and Gellert (5) showed that the decrease in activity with time, after myosin had been treated with P C M B , was accelerated in the presence of substrate. The series of curves obtained (Fig. 6) shows clearly the initial acceleration of ATPase activity and also the decay of activity with
117
increased time of incubation. The "curving off" which occurred before the limit of the linear range had been reached indicates quite adequately the acceleration of the decrease in activity occurring in the presence of ATP. A further application was to the continuous monitoring of a slow transient in ATPase activity. The transient followed was the total dephosphorylation of a small quantity of A T P by a comparatively large quantity of myosin (Fig. 7). For this
Phosphate production with limiting ATP (From 'dye' recording)
0.5
----~
0.2
....... ~.................................. f ' ........................
/
"~
J . 5p3hXslpOh-::p~erS i it e r
o
I
0
l
. . . . .
I.
I
15
30 45 60 Time in minufes FI6.8. Replotting of Fig. 7, together with a comparable curve obtained with the orthophosphate method. ( - - ) The conversion from the dye recording of Fig. 7; (..... ) orthophosphate measurement. Arrows mark the region analyzed by the analogue computer.
OF" P R O D U C T
FORMATION
VS.
TIME
>. t~j . 2 4 1
f
.229
q
.217 .205
I-..193 .181
I
I
I
2t.0 6.0 9.0 T I M E IN M I N U T I ~ S T I M E IN M I N U T E S Fro. 9. The fitting of the slow transient by an analogue computer working on repetitive operation. Left: the recorded transient; right: the oscilloscope trace. 0
118
RAINFORD
purpose the output of the spectrophotometer was fed into a log converter (to change per cent transmission into optical density). The output of the converter was amplified and recorded on a linear scale. The transient obtained followed closely the transient in the same system using the orthophosphate method (Fig. 8). T o illustrate a use of such recordings, the transient was fitted b y an analogue computer, working on repetitive operation, to the differential equation of the scheme: E+S. where
kl k-1
Km
"X
ks --~ E + P,
L I + k~ kl
T h e result obtained (Fig. 9) gave a value of 2.99 X 10-5 M for the constant Kw. This is a very reasonable result for the K~ of myosin, obtained from an experiment at a single substrate concentration. CONCLUSION We feel t h a t the foregoing method is convenient for routine A T P a s e activity determinations. Each run takes up to 5 minutes, and consequently a very large
number of samples can be dealt with in a n y one d a y b y one person. This is in contrast to the m a x i m u m of 18 runs, under the same conditions, using the orthophosphate method or the p H stat. This clear advantage does not count the simplicity in glassware, pipetting, etc. I n addition, by using a rapid mixing chamber, the dye method described should be readily adaptable to recording fast initial transients. ACKNOWLEDGMENTS The author wishes to express her gratitude to Professor Manuel Morales for useful discussion of this work. She is also indebted to Professor I. M. Klotz who kindly gave her the dye, and to Dr. Charles Walter for assistance in obtaining the analogue computer analysis. REFERENCES 1. FISK~, C. H., AND SUEBA Row, Y., J. Biol. Chem. 66, 375 (1925). 2. MARTIN,J. B., AND DOTY, D. M., Anal. Chem. 21,965 (1949). 3. ASAI, H., AND MORALES,M. F., Arch. Biochem. Biophys. 99, 383 (1962). 4. GUTFREUND, ~-L, AND STURTEVANT, J. M., Biochem. J. 63, 656 (1956). 5. GILMOVR,D., ANDGEL~ER%M., Arch. Biochem. Biophys. 93, 605 (1961).