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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 2 of 5 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. 3 of 5 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. 4 of 5 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. 5 of 5