Download Fluorescent Probe Studies of Proteins

DyNA Quant™ Application Note 5
Fluorescent Probe Studies of Proteins
Contributed by Dr. Sonia R. Anderson,
Oregon State University, Department of Biochemistry and Biophysics, Corvallis, Oregon
A
1-Anilinonaphthalene-8-sulfonic
acid (ANS):
Catalytic assays of phosphorylase kinase
A number of fluorescent probes exhibit fluorescence char-
ANS dye is virtually non-fluorescent in water but becomes
acteristics that are easily measured by the fixed-wavelength
highly fluorescent upon binding to such proteins as serum
DyNA Quant 200 Fluorometer, which excites at 365 nm and
albumin, apomyoglobin, apohemoglobin, horse liver alco-
measures emission at 460 nm. These probes include 1-anili-
hol dehydrogenase, calmodulin, and glycogen phosphory-
nonaphthalene-8-sulfonic acid (ANS), 9-anthroylcholine
lase. Depending on conditions, the amount of ANS bound
(9-AC), and 5-dimethylaminonaphthalene-5-sulfonic acid
by the phosphorylated form of glycogen phosphorylase
("dansyl"). Protocols or examples using each of these probes
(phosphorylase a) is at least two to three times greater than
are described in this application note.
that bound by the non-phosphorylated enzyme (phosphorylase b) (1). X-ray crystallographic studies on phosphorylase b show that ANS binds primarily to the activator (AMP)
Section A
Assaying phosphorylase kinase with ANS.
Section B
Assaying protein/peptide binding by calmodulin with dansylcalmodulin.
site, close to the phosphorylated serine residue (SER 14) in
phosphorylase a (2).
Phosphorylase kinase is a calcium-dependent enzyme (containing calmodulin as an integral subunit) that catalyzes the
phosphorylation of glycogen phosphorylase (3, 4).
Section C
Assaying calmodulin binding by smooth muscle myosin light
chain kinase with 9-AC.
Phosphorylase b + ATP
(ANS low fluorescence)
Ca+2
Phosphorylase kinase
Equipment
DyNA Quant 200 Fluorometer
Phosphorylase a + ADP
(ANS high fluorescence)
DQ105 Glass Fluorometry cuvette
80-6237-13
35084/Rev A/6-95
DQ Application Note 5
Fluorescent Probe Studies of
The conversion of phosphorylase b to a has in the past
Assay set up
been monitored using a tedious two-stage procedure
1 To each of two to four cuvettes, add:
involving final colorimetric determination of inorganic
phosphate (5). A more convenient continuous fluoromet-
Phosphorylase b solution (5.2 mg/ml)
ric assay based on the increase in ANS fluorescence is
ATP (0.1 M)
12 µl
described in (6). The rate of change in fluorescence
Calcium acetate (50 mM)
2.4 µl
depends on the phosphorylase kinase concentration and
distilled H2O
also on conditions that affect the enzyme, such as pH and
Use one cuvette to adjust the sensitivity of the fluorometer and to establish the maximum fluorescence of
phosphorylase a.
calcium ion concentration. The ANS dye has little or no
effect on the rate of the phosphorylation reaction.
0.60 ml
0.58 ml
2 To a cuvette containing the above reaction mixture,
Stock buffer
Sodium glycerophosphate
100 mM
Tris base (Adjust to pH 8.3 with HCl)
100 mM
Magnesium acetate
12 mM
Rabbit muscle phosphorylase b
Concentrated stock (>100 mg/ml) in glycerol; prepared
according to procedures in (7).
On the day of the phosphorylase kinase assays, dilute phosphorylase b into the stock buffer for a protein concentration
of 5.2 mg/ml. Dithiothreitol (0.2 M stock) is then added to a
final concentration of 2 mM.
Important: Add a small amount of charcoal (~0.5 mg/ml;
Norit A) to the solution in order to extract residual AMP
from the phosphorylase. Gently swirl, place on ice for 20
minutes, and filter (Millipore 0.45 µm filter) to remove the
charcoal. This step is essential in order to obtain maximum
changes in fluorescence upon phosphorylation.
add a 9.0 µl aliquot of 2 mM ANS and a few µl of the
phosphorylase kinase sample (to give a final concentration in the range of 10 µg/ml). Allow the reaction to
proceed for about 10 minutes.
3 Place the cuvette in the fluorometer cuvette well.
Ensure that the reading is within the instrument’s
range and check the stability of the reading.
4 Remove the cuvette containing the sample and insert a
second cuvette to which neither ANS nor phosphorylase kinase has been added. The second cuvette serves
as a blank. Zero the instrument.
5 Determine the intensity of the first cuvette containing
the reacted sample. (Values in the range of 1000 1500 are recommended.) Save this sample for future
reference. Place it in an area protected from light.
Determining time courses
Figure 1 illustrates several reaction time courses monitored
ATP Solution
on the mini-fluorometer. Reactions were started at
[0.1M ATP]
t = 0 with the addition of phosphorylase kinase. The cap-
Adenosine 5’-triphosphate
0.1 M
tion for Figure 1 describes assay results. Tested parameters
include photochemical decomposition under continued
adjust carefully to pH ~6.7 with 6M KOH
illumination and the effect of phosphorylase kinase and
calcium ion concentrations.
EGTA Solution
[0.2M EGTA]
Ethylene glycol-bis (β-aminoethyl ether)
N,N,N’,N’-tetraacetic acid
0.2 M
adjust to pH 7.0 with 6M KOH
There are two methods for calculating the relative reaction
rates for non-linear time courses. We prefer to calculate the
Calcium acetate
relative slope corresponding to the maximum rate of fluo-
[50mM CaAc2]
CaAc2
Calculating reaction rates
50 mM
rescence change, observed about half way through the
reaction. The relative slope corresponds approximately to
the rate of 32P incorporation determined in radio-assays (6).
ANS Solution
[2mM ANS]
1-Anilinonapthalene-8-sulfonic acid
2 mM
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DQ Application Note 5
Fluorescent Probe Studies of
Note that the rate calculation requires values of F∞ (the
fluorescence obtained with phosphorylase a at equilibrium, Fo (the fluorescence obtained with phosphorylase b at
zero time, and at least four well-spaced intermediate values of F. Approximate rates can be estimated from the halftimes corresponding to 50% of the total fluorescence
change.
Figure 1.
Fluorometric monitoring of phosphorylase conversion
from b to a.
o
•
Control cuvette. (No phosphorylase kinase added). The slight
downward trend illustrates that some photochemical decomposition occurs as a result of illumination.
Enzyme-catalyzed assay containing 5.7 µg/ml phosphorylase
kinase + 0.1 mM Ca2+. Sample was kept in the cuvette well
throughout the reaction time and monitored at intervals.
∇
Enzyme-catalyzed assay containing 5.7 µg/ml phosphorylase
kinase + 0.1 mM Ca2+. Same as , except that the sample was
removed from the cuvette well between readings in order to
minimize photochemical decomposition.
▼
Enzyme-catalyzed reaction containing 2.8 µg/ml phosphorylase kinase + 0.1 mM Ca2+ The sample was removed from the
cuvette well between readings. This assay illustrates the effect
of phosphorylase kinase concentration. (Compare to ∇.)
■
Enzyme-catalyzed reaction containing 2.8 µg/ml phosphorylase kinase. The sample was removed from the cuvette well
between readings. Same as ▼ except that the solution contained 1.0 mM EGTA and no added calcium. This assay illustrates the effect of calcium.
•
Figure 2.
Rate calculation for non-linear time course.
Important notes
➲
Limit sample exposure to fluorescent light. Leave the
sample in the cuvette well only if the reaction is fast
(complete within 4 minutes) and measurements are
made at short intervals (15 seconds).
➲
Add ANS to the phosphorylase solution on the day of
the assay and store the solution in the dark. Generally,
ANS-protein adsorbates are light sensitive and should
not be exposed to any light for prolonged periods.
➲
➲
The recommended ANS concentration (15 µM) is lower
than that originally used by Malencik et al. (6). This
change is necessary in order to stay within the range of
the fluorometer.
Use only fresh glycerophosphate buffers for best results.
Although glycerophosphate has been a "standard"
buffer for phosphorylase kinase assays, more stable TRIS
or MOPS buffers can be used (6).
References
1
Seery, V.L. & S.R. Anderson. (1972) Biochemistry. 11:707-712.
2
Madsen, N.B., S. Shechosky and R.F. Fletterick. (1983)
Biochemistry. 22:4460-4465.
3
Malencik, D.A. and E.H. Fischer. (1983) Calcium and Cell
Function. 4:161-188.
4
Chan, K.F. Jesse and D.J. Graves. (1984) Calcium and Cell
Function. 5:2-31.
5
Krebs, E.G. (1966) Methods Enzymol. 8:543-546.
6
Malencik, D.A., Z. Zhao and S. R. Anderson. (1991) Biochem.
Biophys. Res. Comm. 174:344-350.
7
Fischer, E. H., E. G. Krebs and A. B. Kent. (1958) Biochem. Prep.
6:68-73.
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DQ Application Note 5
Proteins
Fluorescent Probe Studies of
B
Dansyl Calmodulin:
Detection of calmodulin-binding proteins and peptides
Dansyl calmodulin has been used for the identification of
proteins or peptides that interact with calmodulin and for
the determination of binding constants and stoichiometry
(1,2,3). Dansyl calmodulin is an unusually responsive covalent conjugate prepared by labeling calmodulin with
5-(dimethylamino)-1-naphthalenesuflonyl chloride. Both
the fluorescence emission maximum and the quantum
yield of calmodulin change once calcium and proteins or
peptides bind.
The preparation of dansyl calmodulin and its interactions
with myosin light chain kinase and small peptides (1) and
with cyclic nucleotide phosphodiesterase and calcineurin
(2) were independently described in 1982. The spectral
Figure 3.
characteristics of dansyl calmodulin (broad absorption
Fluorescence changes accompanying the stepwise addition of melittin (0.46 mM stock) to 1.6 ml solution containing 2.0 µM dansyl calmodulin in 0.20 M KCl, 20 mM
MOPS, 1 mM CaCl2.
with a maximum near 340 nm) and emission maximum
shifting from 520 nm in the absence of proteins to 480500 nm in the presence of proteins are within the range of
the DyNA Quant 200 Fluorometer. In fact, the wavelength
(460 nm) corresponding to maximum transmission by the
emission filter has been routinely employed to optimize
Comments
the fluorescence change that occurs when dansyl calmod-
Dansyl calmodulin is more light stable than ANS protein-
ulin interacts with proteins and small peptides.
adsorbates. The magnitude of the fluorescence change
may vary, both among calmodulin-binding proteins and
peptides and different preparations of dansyl calmodulin.
Reagents
Dansyl calmodulin (commercially available, Sigma #P6654)
Buffer
Composition may vary. A typical solution contains 0.20 M
KCl, 50 mM MOPS, 1 mM CaCl2 (pH 7.3). The moderately
high ionic strength helps to suppress non-specific interactions.
However, marked enhancement of dansyl calmodulin fluorescence occurs with all known calmodulin-binding proteins and peptides.
References
1
Malencik, D.A. and S.R. Anderson. 1982. Biochemistry
21:3480-3486.
2
Kincaid, R.L, M. Vaughan, J.C. Osborne and V.A. Tkachuk.
1982. J. Biol. Chem. 257:10638-10643.
3
Anderson, S.R. and D.A. Malencik. 1986. Calcium and Cell
Function. VI:1-42.
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DQ Application Note 5
Fluorescent Probe Studies of
C
9-Anthroylcholine:
Probe for Calmodulin and Smooth
Muscle Myosin Light Chain Kinase
9-anthroylcholine (9-AC) is a fluorescent dye resembling
1-anilinonaphthalene-8-sulfonate (ANS) in its ability to
undergo large increases in quantum yield upon binding to
specific proteins. However, 9-AC is probably more selective
than ANS. It undergoes a relatively low-affinity calciumdependent interaction with calmodulin (1) and, in addition, a higher affinity calmodulin-dependent interaction
with smooth muscle myosin light chain kinase (2, 3).
Results shown in Figure 4 illustrate these associations.
Interestingly, 9-AC is specific for smooth muscle myosin
light chain kinase (MLCK). (It does not bind to the related
enzyme, skeletal muscle myosin light chain kinase.) The
fluorescence measurements were taken in the presence of
Figure 4.
calmodulin and 9-AC as a means of identifying smooth
9-Anthroylcholine: Probe of calmodulin and smooth
muscle myosin light chain kinase.
muscle MLCK during purification procedures.
Reagents
o
Calmodulin added to a solution of 5 µM 9-AC + 1 mM EGTA.
•
Calmodulin added to a solution of 5 µM 9-AC + 1 mM CaCl2.
9-Anthroylcholine
(commercially available solid form),
5.0 µM
∇
Calmodulin added to a solution of 5 µM 9-AC + smooth muscle myosin light chain kinase (0.35 µM) +1 mM EGTA.
MOPS, pH 7.3
50 mM
▼
Calmodulin added to a solution of 5 µM 9-AC + smooth muscle myosin light chain kinase (0.35 µM) + 1 mM CaCl2.
KCl
CaCl2 or EGTA
0.10 M
1 mM
Important notes
➲
Avoid prolonged illumination of solutions containing
9-AC.
➲
9-AC undergoes gradual hydrolysis. Therefore stock
solutions of 9-AC (1 to 2 mM) should be frozen and
stored for no longer than one month.
References
1
LaPorte, D.C., B.M. Wierman and D.R. Storm. (1980)
Biochemistry. 19:3814.
2
Malencik, D.A., S.R. Anderson, J.L. Bohnert and Y. Shalitin.
(1982) Biochemistry. 21:4031-4039.
3
Malencik, D.A. and S.R. Anderson. (1986) Biochemistry.
25:709.
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