Vol. 46, No. 2
Printed in U.S.A.
T U B AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1906 by The Williams & Wilkins Co.
CREATINE PHOSPHOKINASE DETERMINATION BY THE
NINHYDRIN REACTION
FLUORESCENT
REX B. CONN, J B . , M.D., AND VICENTE ANIDO, M.D.
Department of Pathology, West Virginia University, Morgantown, West Virginia
Creatine phosphokinase (CPK) (adenosine triphosphate [ATP] creatine transphosphorylase) catalyzes the reaction:
Creatine + ATP <=* Creatine-P0 4 + ADP
The enzyme is present in large quantities in
skeletal and heart muscle, and measurement of its serum levels has proven valuable
in the diagnosis of myocardial infarction
and certain types of muscular dystrophy. 6 ' 7| 8 It has a definite advantage
over other enzyme tests used for this purpose in that neither pulmonary disease nor
hepatic disease produce elevated serum
levels of CPK.
Several methods for determining CPK
have been proposed. These methods are
based upon 1 or another of the following
principles:
A. Measurement of creatine phosphate
(CP) formation in the forward reaction
by the determination of "apparent" inorganic phosphate in CP with a colorimetric method.10
B. Measurement of ATP produced in the
reverse reaction by utilization of it in ahexokinase-glucose-6-phosphate dehydrogenase
coupled reaction. The reaction rate is followed by spectrophotometric measurement
of nicotinamide adenine dinucleotide phosphate (NADPH) production.12
Glucose + ATP *± Glucose-6-P0 4 + ADP
Glucosc-6-P0 4 + NADP+
<=* 6-phosphogluconate + NADPH + H+
C. Measurement of adenosine diphosphate (ADP) production in the forward
reaction by a coupled pyruvate kinaselactate dehydrogenase system which can be
followed spectrophotometrically by measurReceived, January 31, 1966.
This investigation was supported in whole by
Public Health Research Grants R01 AM 09372-01
MET from The National Institute of Arthritis
and Metabolic Diseases and R01 12884-01 from
The National Institute of General Medical Sciences.
ing the disappearance of nicotinamide
adenine dinucleotide (NADH). 15
Phosphoenolpyruvate + ADP
<=* Pyruvate + ATP
Pyruvate + NADH + H+
<=± Lactate + NAD+
D. Measurement of creatine produced
in the reverse reaction by means of the
diacetyl reaction.1, "• 6 ' 8
The kinetics of creatine phosphokinase
are complex, and there is disagreement on
optimal substrate concentrations, product
inhibition, activation by sulfhydryl groups,
anion inhibition, and on what constitutes
the "true" substrate of the enzyme. In
addition, a much higher concentration of
enzyme is apparently needed to catalyze
the forward reaction than the reverse reaction at comparable rates.
Because of the apparent clinical usefulness of the CPK determination, we wished
to study some of the problems related to
its measurement and to adapt a previously
described fluorometric method for measuring
creatine 2,3 to the determination of CPK.
Utilization of the reverse reaction appeared
most suitable, first because a given enzyme
concentration produces a greater activity
in this direction,6 and second, because utilization of the forward reaction would require measurement of a small change in a
relatively large creatine concentration,
rather than the production of small quantities of creatine, as would take place in the
reverse reaction. In our experience the
fluorescent ninhydrin reaction has proved
more useful for the measurement of creatine
than has the diacetyl reaction. Sax and
Moore14 have recently reported the use of
the fluoresccnt-ninhydrin reaction for assay of CPK activity. Our results are in
agreement with theirs, although the conditions selected in the present report resulted
in a higher enzymatic activity and we believe that they represent more nearly
177
178
CONN AND ANIDO
optimal conditions for the determination of
CPK.
METHODS
A series of experiments was made, to
determine the optimal conditions for measurement of CPK activity. Normal human
serum contains only small amounts of CPK,
and it was more convenient in these experiments to use either rabbit serum or purified
rabbit CPK dissolved in buffer. Critical
results were confirmed with human serum.
The final concentrations used in the incubation mixture were: Tris buffer, 0.05 M;
magnesium, 0.02 M; Cysteine, 0.01 M;
CP, 0.02 M; and ADP, 0.02 M...The volume
of the incubation mixture selected for routine use was 1.0 ml., including 0.1 ml. of
serum. A 10-min. incubation period at pH
6.8 and 37 C. produced optimal results. A
separate serum blank containing all of the
above ingredients except ADP, for which
water was substituted, was included for
each specimen to compensate for any
creatine present originally in the serum,
added in the CP, or formed by nonenzymatic hydrolysis. After incubation, creatine
was measured by the fluorescent-ninhydrin
method. This method has proved to be
simple and sensitive and to have very good
precision. A Ba(OH) 2 -ZnS04 filtrate is
made, and an aliquot of filtrate is treated
with ninhydrin in a highly alkaline solution.
The resulting fluorescence is read at 525
in/* with an excitation beam at 410 my.. A
slight modification was necessary to adapt
the reaction for enzyme assay. The high
magnesium concentration in the buffer
interfered with the fluorescent reaction.
This problem was eliminated by chelating
the magnesium with ethylenediaminetetraacetate (EDTA), which in turn required a
change in concentration of the Ba(OH) 2
used in protein precipitation.
REAGENTS
All reagents are prepared with deionized,
glass-distilled water.
Tris (0.1 M) magnesium (0.04 M) buffer.
Dissolve 1.21 Gm. of tris (hydroxymethyl)
aminomethane, buffer grade, and 0.S6 Gm.
of magnesium acetate, Mg(CH 3 COO) 2 -4H 2 0
Vol. 46
in approximately 60.0 ml. of water. Adjust
the pH to 6.8, using 0.1 N acetic acid. Dilute
to 100 ml. This solution is stable but the
pH should be checked occasionally.
Cysteine (0.05 M). Dissolve O.OSS Gm. of
cysteine (L-cysteine hydrochloride • H 2 0) in
approximately 6.0 ml. of water; adjust the
pH to 6.8, using 0.1 N NaOH; and dilute
to 10 ml. with water. Cysteine deteriorates
rapidly and it is essential that this solution
be made up immediately before use. Attempts to preserve cysteine solutions by
freezing were not completely successful,
and freshly-prepared solutions are recommended.
Creatine phosphate (0.2 M). Dissolve
0.654 Gm. of CP (creatine phosphate,
sodium salt hydrate) in 8.0 ml. of water.
Adjust the pH to S.0, using glacial acetic
acid. (There is some breakdown of creatine
phosphate if it is stored at lower pH.) Dilute
to 10 ml. and freeze in small aliquots in
tightly stoppered tubes.
ADP solution (0.2 M). Dissolve 0.S9S
Gm. of ADP (adenosine-5'-diphosphate,
sodium salt) in 8.0 ml. of water. Adjust pH
to 6.S, using 0.1 N NaOH. Dilute to 10.0
ml. and freeze in small aliquots in tightly
stoppered tubes.
EDTA solution (0.00314 M). Dissolve
1.17 Gm. of disodium EDTA in 1 liter of
deionized water. This is stable at room temperature.
Zinc sulfate (5 per cent iv/v). Dissolve
50 Gm. of zinc sulfate (ZnS0 4 -7H 2 0) in 1
liter of deionized water. This is stable at
room temperature.
Barium hydroxide (0.35 N). Dissolve
55.0 Gm. of barium hydroxide (Ba(OH) 2 8H 2 0) in 1 liter of water and store at room
temperature in a buret protected by sodalime tubes. The Ba(OH) 2 is more concentrated, because of the EDTA, than in the
Somogyi filtrate; however, the procedure
for balancing the Ba(OH) 2 and Z11SO4 is
similar. To titrate mix 1.0 ml. of ZnS04
with 7.0 ml. of EDTA, 0.7 ml. of buffer,
0.3 ml. of water, and 4 drops of phenolphthalein. Add Ba(OH) 2 in drops, with
continual agitation (1.0 ml. of Ba(OH) 2
should exactly neutralize the mixture).
Adjust the Ba(OH) 2 or Z11SO4 concentration if necessary.
Aug. 1966
179
FLUOROMETRIC CPK ASSAY
Ninhydrin (1.0 per cent w/v alcoholic).
Dissolve 1.0 Gm. of ninhydrin ( 1 , 2 , 3 , triketohydrindene • H 2 0) in 100 ml. of 95
per cent ethyl alcohol. This is stable for
approximately 1 week in the refrigerator.
Potassium hydroxide (10 per cent w/v).
Dissolve 100 Gm. of potassium hydroxide
in 1000 ml. of deionized water. Filter and
store at room temperature.
Quinine standard. Dissolve 2 mg. of
quinine sulfate in 1 liter of 0.1 N H2SO4.
Store at room temperature. This is a stable
fluorescent standard used for calibration
of the fluorometer, and the exact concentration is not critical.
Creatine standards, (a). Stock standard
(10 mM. per liter). Dissolve 0.1492 Gm. of
creatine monohydrate in 100 ml. of water.
Freeze in small aliquots. (b). Working
standard (0.01 mM. per liter). Dilute the
stock creatine standard 1:1000. Prepare a
fresl/solution every day or 2.
PROCEDURE
1. For each specimen and control, prepare
2 tubes containing: Tris-Mg buffer, 0.5 ml.;
cysteine, 0.2 ml.; CP, 0.1 ml.; and serum,
0.1 ml.
2. Bring all tubes to 37 C. by placing
them in a water-bath for a few minutes.
3. To start the reaction, add 0.1 ml. of
ADP to 1 tube. Add 0.1 ml. of water to
the other tube for the blank.
4. Incubate for 10 min. at 37 C.
5. Prepare filtrates by adding to each
tube 7.0 ml. of EDTA, 1.0 ml. of Ba(OH) 2
(mix well), and 1.0 ml. of ZnSCi.
6. Filter through Whatman No. 1 filter
paper (or centrifuge at high speed for 5
min.).
7. Pipet into cuvets 2.0 ml. of filtrate
(or supernatant), 1.0 ml. of ninhydrin, and
1.0 ml. of KOH. Standards and a standard
blank are also prepared by using 2.0 ml. of
standard and 2.0 ml. of water, respectively,
instead of filtrate.
S. Take fluorometric readings exactly 5
min. after the addition of KOH.
9. Calcidaliojis. Subtract serum blank
reading from unknown reading. Subtract
standard blank from standard.
Reading of unknown
Reading standard
y
10 nM. per liter
1000
10 ml.
1
10 min.
0.1 ml.
= pM of creatine formed per min. per ml.
Units per ml. of serum
= juM per min. per ml. X 1000
Fluorometry
Fluorescence was measured with a Photovolt Fluorometer consisting of a lineoperated Multiplier Photometer Model
520-M and Fluorescence Unit Model 54.
The excitation beam at 410 mn was isolated with Corning glass filters No. 3S50
and No. 5S50 used together. The emission
beam at 525 mji was isolated with Corning
glass filters No. 33S5 and No. 97S2 used
together. Other photofluorometers and spectrophotofluorometers have also been found
to be satisfactory. With the reagents described, fluorescence reaches a maximum
at 5 min. after addition of KOH and then
declines slowly. Because of this instability
of the fluorescent compound, it is convenient
to use the stable quinine standard to set
the instrument at a convenient sensitivity,
and then settings may be verified whenever
this is desired. The readings for the quinine
standard are not used in any calculations.
RESULTS AND DISCUSSION
Several factors combine to make the
determination of CPK somewhat more
exacting than many of the enzyme assays
performed routinely in the clinical laboratory. Labile, expensive substrates require
more than routine care in preparation. The
enzyme itself is inhibited by numerous substances, and scrupulously clean glassware
is a prerequisite for reliable results. All
reagents should be prepared with deionized,
glass-distilled water, to avoid possible
heavy-metal contamination. I t is also recommended that suitable control solutions
(rabbit serum is satisfactory) be included
with each batch, and that a reagent blank
as well as a blank for each unknown be run.
Duma and Siegel5 and Sax and Moore14
180
Vol. 46
CONN AND ANIDO
recommend omission of the serum blank.
Although creatine in serum is sufficiently
low that it would not cause a significant
error in a sample with a grossly elevated
CPK activity, it may produce an error of
10 to 20 per cent in the normal range. The
serum blank also provides a valuable check
on technic and reagents.
Selection of Buffer
The prerequisites for buffer selection
were negligible inherent fluorescence, absence of interference in the fluorescent
ninhydrin reaction, and absence of interference in the enzyme system. The first 2
conditions are easily evaluated. To evaluate
the third condition, different buffers were
set up in comparable incubation mixtures,
and the activity of purified CPK was assayed with each buffer. Numerous compounds and ions have an inhibitory effect
upon CPK, and an empiric approach to
buffer selection was necessary. The buffer
resulting in the highest measured enzyme
activity was considered to be most suitable.
Tris buffer resulted in highest activity.
Acetic acid, rather than hydrochloric acid,
is used to adjust the pH to avoid inhibition
by chloride ions. The buffer concentration
may be varied over a range of 0.02 M to
0.20 M without appreciable effect on the
reaction rate under the conditions described.
Effect of pH
The optimal pH for the forward and reverse reactions differs appreciably.11 In the
reverse reaction, as used in the present
experiments, the optimal pH was found to
be 6.8. This was the pH selected for use,
although in certain applications it might not
be the most satisfactory. When the pH of
the incubation mixture is below 7.0, the
spontaneous breakdown of CP is accelerated. Inclusion of a blank compensates
for this; however, the sensitivity of the
assay is decreased because of the difficulty
in measuring very small changes in creatine
concentration in the presence of larger
quantities of creatine in the serum, in the
creatine phosphate, and in that formed by
nonenzymatic hydrolysis. Even with the
low CPK activities found in normal serum,
however, these factors were never great
enough to produce appreciable interference.
Effect of Temperature and Incubation Time
Two incubation temperatures were evaluated, 25 C. and 37 C. The latter was
chosen because it resulted in greater enzymatic activity under the conditions selected. The 10-min. incubation period was
selected for convenience. Product formation
was found to be directly related to time of
incubation if the substrate concentration
remains sufficiently high to maintain zeroorder kinetics.
Substrate Concentrations
A series of cross-experiments was performed, in which the concentrations of CP,
ADP, and Mg were varied independently.
Because of the inhibitory effects of chloride
and sulfate ions,13 magnesium diacetate
was used to supply magnesium ions. I t was
found that at the concentrations used, the
optimal concentration of each substrate
was not independent of the others. Figures
1 and 2 illustrate the concentration-activity
curves obtained for CP and ADP, respectively. The inhibitory effect of high ADP
concentrations noted by other investigators
was not seen under the conditions selected,
although a marked inhibitory effect was
present under other conditions of incubation.
The reason for this variable effect is not
clear and is under further study. The concentrations of CP and ADP selected are
higher than those used in other described
methods. Chappell and Perry 1 used 0.004
M CP, 0.002 M ADP, and 0.005 M Mg. Sax
and Moore16 used 0.0075 M CP, 0.003 M
ADP, and 0.010 M Mg. Earlier workers used
lower concentrations of CP and ADP. In
view of the rather low CPK activity in
normal serums, it appears to be advisable
to use substrate concentrations which produce optimal activity. The higher substrate
concentrations also seem to permit measurement of greater CPK activities, while
maintaining zero-order kinetics.
Effect of Cysteine
In agreement with Ennor and Rosenberg, 6
cysteine was found to have a negligible
Aw/. 1966
181
FLTJOROMETR10 CPK ASSAY
effect on the forward reaction, but its effect
on the i'everse reaction was to enhance the
measured activity as much as 100 per cent.
In the concentrations used (after protein
precipitation), cysteine had no inherent
fluorescence and did not interfere with
measurement of creatine. In very high
concentrations cysteine reacted with ninnydrin to give a blue color, with subsequent
interference in the fluorometric measure-
0.02
Q03
0.04
CREATINE PHOSPHATE ( M O I E S / I . )
FIG. 1. The effect of varying concentrations of creatine phosphate on
creatine phosphokinase activity. Concentrations of other constituents
are as described in the text.
0.01
0.02
ADP
003
0.04
(MOIES/I.)
FIG. 2. The effect of varying concentrations of adenosine diphosphate
on creatine phosphokinase activity. Concentrations of other constituents
are as described in the text.
182
CONN AND ANIDO
ments. In addition to enhancing CPK acactivity, sulfhydryl compounds also appeared to make time curves more linear
and results more reproducible.
Measurement of Creatine Formed
High concentrations of magnesium were
found to interfere seriously with the measurement of creatine by the fluorometric technic.
This interference apparently is due to
destruction of an intermediate compound,
o-carboxyphenylgloxal,3 rather than to
quenching of fluorescence. The concentration of magnesium, after the dilution which
occurs during protein precipitation, is low
enough so that this interference is not great;
however, the addition of EDTA completely
eliminates such interference. EDTA itself has negligible fluorescence at the wave
lengths used, and does not interfere with
measurement of creatine. In the method
outlined, a 10 per cent excess of EDTA is
used. An additional advantage of using
EDTA is that removal of Mg++ ions stops
enzymatic activity. Although we were unable to confirm it, Chappell and Perry 1 have
reported that residual CPK activity may
be present in Ba(OH) 2 -ZnS04 filtrates.
None of the other constituents in the
incubation mixture was found to interfere
with measurement of creatine. CP does not
react with ninhydrin in the fluorogenic
reaction, and it is stable in alkaline solutions. Large amounts of free creatine in
the creatine phosphate would tend to reduce the sensitivity of the method, but would
introduce no error, since the inclusion of CP
in the blank compensates for any free
creatine. The same factors hold true for the
presence of creatine in the serum samples.
The amount of free creatine in CP obtained
from some sources was very high. CP from
Sigma Chemical Co. (St. Louis, Missouri)
produced consistently low reagent blanks.
Previous experiments have shown that
the fluorescent ninhydrin reaction takes
place equally well in aqueous and in alcoholic solutions, although the fluorescence
produced is somewhat greater in the latter.
In the present method aqueous KOH is
used because of ease in preparation, but
there is a slight sacrifice hi the degree of
fluorescence produced.
Vol. 46
Creatine for preparation of standards
may be recrystallized by the method of
Hunter; 9 however, several lots of creatine
hydrate from Eastman Organic Chemicals
(Rochester, New York) have been tested,
and recrystallization was not found to be
necessary.
Although the diacetyl reaction has been
used in several colorimetric procedures for
measuring CPK, sulfhydryl groups seriously
interfere with this reaction and it is not
entirely suitable for CPK assay. The groups'
inhibitory effect has been decreased or removed by treatment with /j-chloromercuribenzoate and increased in the tune allowed
for color development to 60 min.8 We have
found no interference by sulfhydryl groups
in the fluorescent reaction described above.
In very high concentrations they will produce a quenching effect, and cysteine in very
high concentrations will produce a blue color
in the cuvet.
Linearity and Precision of the Method
The limits of linearity for the fluorometric CPK assay are determined at the
lower end by the minimal amount of creatine
which can be measured accurately in the
presence of the free creatine introduced
with the CP and serum and by nonenzymatic hydrolysis. The upper limit is determined by the maximal product formation
or substrate utilization that can occur
without a change in reaction rate. An additional consideration at the upper limit
is the linearity of the fluorogenic reaction
which is subject to concentration quenching.
The latter was found to be linear to a concentration of creatine which would represent a serum CPK activity of 1.5 JX moles
per min. per ml. of serum (1500 units per
ml.). Higher concentrations of creatine can
be measured accurately by merely diluting
the Ba(OH)2-ZnSO-4 filtrate before carrying
out the fluorogenic reaction. Linearity of
the enzyme reaction itself was assessed by
measuring the activities of serial dilutions
of a highly active rabbit muscle CPK preparation. The reaction produced linear results
to an activity corresponding to 2.0 /mioles
per min. per ml. of serum (2000 units per
ml.). This represented utilization of 10 per
cent of the substrate during the 10-min.
Aug. 1966
183
FLUOBOMETKIC CPK ASSAY
TABLE 1
NORMAL VALUES FOB SEBUM CREATINE PIIOSPHOKINASE*
Men
Fluorescent ninliydrin (this report)
Fluorescent ninliydrin14
Diacctyl 8
Spectrophotonietric12
Spectrophotometries
Spectrophotonietric7
Reverse
Reverse
Reverse
Reverse
Forward
Forward
Women
Mean
Range ± 2<r
Mean
Range ± 2<r
73.8
27.2
40.2
6.4
28.7-189.7
13-57
5.5-74.7
2.3-17.7
58.4
25.8
27.0
3.8
31.5-108.7
13-52
14.3-39.7
0.9-16.0
ot
0.8J
* All values have been converted to units used in the present method. Units per ml. of serum
per min. per ml. X 1000.
f Sex not specified.
| No sex difference.
period, and with slightly greater enzyme
concentrations the reaction became nonlinear. In the method described, if the
enzyme activity of a serum specimen is
greater than 1500 units per ml., a dilution
of the nitrate is necessary. If the activity is
greater than 2000 units, the entire procedure should be repeated with a dilution
(in buffer) of the original serum.
To determine precision of the method,
the coefficient of variation was calculated
from 35 duplicate determinations on human
serums with CPIv values within the normal
range. The coefficient of variation was
5.2 per cent. Precision was also assessed on
a frozen human serum pool. Thirteen determinations on the pool carried out over a
12-day period had a coefficient of variation
of 6.2 per cent. Our data suggest some instability of CPK in frozen serum when it is
stored for periods longer than 2 weeks.
Normal Values
Table 1 lists the normal range for 30
male and 31 female subjects selected from
the laboratory staff and blood donors. The
data for both groups was skewed; however,
a log transformation gave a normal distribution, and the means and 95 per cent
normal ranges are calculated from the transformed data. For purposes of comparison,
the normal values determined by several
other investigators are included. Normal
values from earlier publications are considerably lower than those presented.12
i&l
Where it is possible, the originator of the
method is quoted, and publications giving
normal values on patients are excluded. I t
may be seen that much lower values are
obtained by methods using the forward
reaction, which is to be expected from the
behavior of the enzyme.6 A significant difference in CPIv values between sexes was noted
when methods employing the reverse reaction were used, whereas no such difference
was seen if the forward reaction was used.
Men appear to have not only higher CPK
values than women, but also a greater
dispersion of values. The marked differences
in normal ranges obtained by different
methods appear to be a function of the
incubation conditions selected. Our own
values are higher than other published
values, and we believe that this is a reflection of more optimal conditions of incubation. I t is clear that whenever results of
CPK measurement are reported or published it is imperative that the conditions
of assay be precisely defined.
SUMMARY
A method for the assay of serum creatine
piiosphokinase (CPK) activity which is
based upon a previously described fluorometric method for measurement of creatine
is described. The effects of variation in
conditions of incubation are described, and
published normal values obtained by means
of different methods are compared. The
present method gives higher normal values
184
CONN AND ANIDO
for serum CPK activity, which seem to be
due to more optimal conditions of incubation. The fluorometric method for measuring
creatine in the CPK assay appears to be
superior to the diacetyl method previously
used.
Acknowledgments. Mrs. Beverly Barnett, M.T.
(ASCP), and Mrs. Gerda Carney rendered technical assistance, and Dr. Roger Flora gave advice
pertaining to statistical analysis.
REFERENCES
1. Chappell, J. B., and Perry, S. V.: Creatine
phosphokinase: assay and application for
the micro-determination of the adenine nucleotides. Biochem. J., 57: 421-427, 1954.
2. Conn, R. B., Jr.: Fluorimetric determination
of creatine. Clin. Chem., 6: 537-548, 19G0.
3. Conn, R. B., Jr., and Davis, R. B.: Green
fluorescence of guanidinium compounds with
ninhydrin. Nature, 1SS: 1053-1055, 1959.
4. Dreyfus, J. C , and Schapira, G.: Technique
de dosage de la creatine phosphokinase du
serum. Rev. frang. etud. clin. biol., 6: 700703, 1901.
5. Duma, R. J., and Siegel, A. L.: Serum creatinine phosphokinase in acute myocardial
infarction. Arch. Intern. Med., 115: 443451, 1965.
6. Ennor, A. H., and Rosenberg, IT.: Some properties of creatine phosphokinase. Biochem. J., 57: 203-212, 1954.
Vol. 46
7. Hess, J. W., MacDonald, R. P., Frederick, R.
J., Jones, R. N., Neely, J., and Gross, D.:
Serum creatine phosphokinase (CPK) activity in disorders of heart and skeletal
muscle. Ann. Intern. Med., 61: 1015-1028,
1964.
8. Hughes, B. P.: A method for the estimation of
serum creatine kinase and its use in comparing creatine kinase and aldolase activity
in normal and pathological sera. Clin.
Chim. Acta, 7: 597-603, 1902.
9. Hunter, A.: Creatine and Creatinine. London: Longmans Green and Co., Ltd., 1928,
p. 54.
10. Kuby, S. A., Noda, L., and Lardy, H.: Adenosinetriphosphate-creatine transphosphorylase. J. Biol. Chem., 209: 191-201, 1954. '
11. Kuby, S. A., and Noltmann, E. A.: ATPcreatine transphosphorylase. In Boyer, P.
D., Lardy, H., and Myrbiick, R.: The Enzymes, Vol. 6. New York: Academic Press,
Inc., 1962, pp. 515-603.
12. Nielsen, L., and Ludvigsen, B.: Improved
method for determination of creatine kinase.
J. Lab. & Clin. Med., 62: 159-168, 1963.
13. Nihei, T., Noda, L., and Morales, M. F.:
Kinetic properties and equilibrium constant
of the adenosine triphosphate-creatine
transphosphorylase-catalyzed reaction. J.
Biol. Chem., 236: 3203-3209, 1961.
14. Sax, S. M., and Moore, J. J.: Fluorometric
measurement of creatine kinase activity.
Clin. Chem., 11: 951-958, 1965.
15. Tanzer, M. L., and Gilvarg, C.: Creatine and
creatine kinase measurement. J. Biol.
Chem., 234: 3201-3204, 1959.