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M. A. Carper and W. R. Carper1 California State College
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Los Angeles, 90032
Biochemistry Laboratory Experiment Polarized fluorescence
The establishment of simultaneous and consecutive interactions between the enzyme site, coenzyme, substrate, and inhibitors has proved to be a difficult problem for all biochemists. I n addition, many limitations in equipment sensitivity to the complexes formed between these compounds, make verification of reaction mechanisms such as (1-5) somewhat difficult.
TPNH + [DPNH]
*?
DPN I + [TPN]
?
where E = enzyme, C = ternary complex, S = substrate, Sox = oxidized substrate, and I = inhibitor. By polarized fluorescence spectroscopy these complexes can be detected at very low concentrations, with apparent speed and ease (1). As Vellick (1) and Albrecht (2) point out, fluorescence is a common phenomena resulting from the competing processes of light emission and non-radiational energy loss. Due to the dependence of fluorescence intensit,y on environmental conditions, fluorescence is a sensitive indicator for chemical interactions. Complex formation may either diminish or enhance the fluorescence intensity as Vellick ( 1 ) shows with the binding of DPN to glyceraldehyde-3-phosphate and lactic dehydrogenase. Furthermore, the change in fluorescence polarization is a result of complex formation, and the polarization factor increases as a function of the mole fraction of the bound form. A more detailed explanation is as follows. The development of this effect involves the rate of decay of fluorescence and thus the lifetime of the excited state. The average lifetime of the excited stat,e, 7 , is the reciprocal of the first-order rate constant. Small molecules in a viscous solvent or large molecules in a solvent of low viscosity exhibit limited motion during the lifetime of the excited state. If these same molecules are excited with plane polarized light, the resulting fluorescence will be polarized with its electric vector usually parallel t.o that of the activating beam. Small molecules in a solvent of low viscosit,y assumc a nearly random orientation during the interval, 7 , and the fluorescence is largely depolarized. However, if the small molecule forms a complex with a large molecule; Present address: Wichita State University, Wichita, Kansas, 67208.
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the fluorescence polarization of the small molecule will change as its rate of tumbling has been decreased. The polarization factor (P) is given by eqn. (6)
where Fl; (=FEE)is the fluorescence intensity measured with the polarizing prisms oriented with electric vectors parallel. F , (=FEa) is the intensity measured with is a correction the prisms crossed. The ratio FRE/FBB factor which accounts for the selective transmission of the emission monochromator (B polarized). If this ratio were not involved thc observed spectrum would be shifted approximately 2 mp to longer wavelengths (3). Glucose dehydrogenase (GDH) is a typical DPN requiring a coenzyme system, and demonstrates quite readily the enzyme-DPN complex. The enzyme catalyzes reaction (7) Glucose + Enayme DPN s Complex
+
-+
glueonolactone
E
+ DPNH
(7)
Pyridoxamine, a fluorescing compound, is also known to inhibit reaction (4) and can be used to identify some of the complexes formed. Experimental
Materials: Pyridoxamine and NAD were obtained from the California Biochemical Co. Reactions were all run in 0.05 M potassium phosphate buffer at p H 7.6. Enzyme: Bovine glucose dehydrogenase (GDH) was purified by the method of Strecker (5) through the second calcium phosphate gel treatment and concentrated as described. This enzyme preparation had a 280 mr/260 mp ratio of 1.47 and a specific activity in excess of 1000. Highly purified enzymes are required for all polarization experiments. With the many improvements in isolation and purification procedures, more enzymes are readily available through commercial sources a t lower prices. Enzymes such as alcohol dehydrogenase, pyruvate kinase, aldolase, catalase, and enolase can now be easily obtained. Fluorometer and Polarization: Fluorescence measurements were made with an Aminco Bowman spectrofluorometer. Slits allowing a 20 mp hand pass were used with an RCA 1B-28 photomultiplier tube. For polarization work the Aminco Bowman spectrofluorometer was equipped with Glan-Thompson prisms mounted directly in front of the entrance and exit slits of the cell holder. The fluorescence is measured with prisms in all four possible permutations. The temperature during all experiments mas main-
tained at 25 + O.l°C by use of liquid cooled cell compartments coupled with a Sargent circulating constant temperature bath. Procedure: Polarization was followed by means of fluorometric titrations in which various concentrations of titrant (non-fluorescing compound) were added to separate portions of test solutions (fluorescing compound). Between additions, solutions are incubated for 5 min a t 25'C, and fluorescence is then measured with prisms in all four positions. In all titrations a fluorescing compound is titrated with a non-fluorescing compound. In some cases displacement can he followed hy addition of a fluorescing compound to a solution containing a known complex which consists of a fluorescing and a non-fluorescing species. By taking readings at two wavelengths, both polarization increase and decrease can be followed. Some compounds can serve as both fluorescing and non-fluorescing depending upon the wavelength chosen. If activated a t 340 mr, glucose dehydrogenase (GDH) does not fluoresce: however, if it is activated at 270 mr, a strong fluorescehce peak i i obtained.
Figure 1. Quenching of fluorescence (open circler) and change in polorimtion (fllled cir&s) when pyridoxamine is titrokd with DPN. Excitation = 325 mp, emission = 390 mp.
Results and Discussion
I n Figure 1 one can readily see the simultaneous increase in polarization (P) and quenching of fluorescence when pyridoxamine is titrated with DPN. This result clearly verifies the existence of an inhibitor-coenzyme complex which the kineticist must take into consideration (4). In addition to eqn. (5), some of the complexes that can be studied (4) are
+
Pyridoxamine GIIII [Pyridoxarnine.GDH] [Pyridoxamine .GDH] DPN DPNH
+
73 ?
+ DPN + ?
+ DPNH * ? 73 ?
(8) (9)
(10) (11)
Figure 2 contains the results of two experiments. First of all the inhibitor, pyridoxamine, was titrated
!
02
["ADPO
.a 0
1
.2
3
h l GOY ~ ADMD
4
5
d
DDH] ccnsrarrr 05%G TOTAL
0-7
Cbcj w a ~ ~r r urra, a
Figwe 2. Changes in fluorescence polorirotion of pyridoxamine during litrotion wilh GDH followed b y rtepwire addition of DPN. lnitiol concentration of pyridaiomine war 4.55 X 10-'M in 0.05 M potorrium phosphate buffer, pH 7.6. Excitation = 325 mp, emirtion = 390 m p
with the enzyme (GDH) to establish the validity of eqn. (2). This was subsequently followed by the addition of DPN which apparently displaces the inhibitor from the enzyme. Figure 3 is the result of a typical competition experiment in which one follows the changes in polarization for two species simultaneously. In this experiment the GDH-pyridoxamine complex was titrated with DPNH and the net result was the breakup of the enzyme-inhibitor complex. In0 terestingly enough, the simultaneous .'aO 3 maximumandmini8 mum in the polari6 zation plot occurs at the approximate enzyme concentration, thus giving another example of the law of contin1 . 0 lo7 1CS uous variation. Ilro* , * m r r n r # r r * As a closing Figure 3. Chmger in fluorescence polorizastatement, it is imtion of pyridoxomine (circle4 and DPNH portant to point (triangles) during titrotion of the GDHpyridoxomine complex with DPNH. lnitiol out t.hat although complex consisted of 3.3 X lo-' M pyripositive results are dorarnine plus 0.55 mg of G D H in 1.5 mi of indicative of com0.05 M potosrium phosphate buffer pH 7.6. For pyridoxomine, excitation = 325 mp, plex formation, emis,ion = 390 m p For DPNH, excitation negative results do = 350 mp, emision= 450 mw not guarantee a lack of complexation. Despite this note of caution, however, it is readily apparent that both student and instructor can obtain a great deal of useful information by means of polarization studies such as those described in this report. Acknowledgment
The authors wish to acknowledge financial support from the National Science Foundation Grant GP-6031. The editors of Archives of Biochemistry and Biophysics were kind enough to grant permission for the republication of the figures utilized in this text. Volume 45, Number 10, October 1968
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Literature Cited ( 1 ) VELICK,S. F., J . Biol. Chem., 233,1455, (1954). ( 2 ) ALBRECHT, A. C., J . Mol. Spec., 6 , 8 4 (1961). ( 3 ) AZUMI,T., AND MCGLYNN, S. P., J. Chem. Phys., 37, 2413, (1962).
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(4)
PAULE, M. R., ANDREOLI, A. J., CARPER, M. A,, A N D CARPER,
W . R., Arch. Bioehem. ond Biophys, 123.9 (1968). (5) STRECKER, H. J. in "Methods in Enzymology," Vol. I (44), (Editors: COJBWICK, S. P., AND KAPLAN,N. O.), Amdemie Press, Ine., New York, 1955, p. 335.
