Salvatore F. Russo'
Biochemistry Department University of Washington Seattle
TWO Fluorescence Experiments
In the last few years it has become more important for students in biochemistry and allied fields to take a course in physical biochemistry. This is necessitated by the fact that biochemistry is increasingly relying on physical measurements to characterize the properties of biological materials. I n the spring of 1967 an experimental laboratory course was initiated under the direction of Dr. David Teller. Some of the experiments done included ultracentrifugation, electrophoresis, difference absorption spectra, and fluorescence. Biochemistry and related fields have increasingly called upon fluorescence for analysis and as a research tool ( I , 8). A typical example is the determination of phenylalanine in blood, an important diagnosis of the hereditary disorder phenylketonuria. I n 1962 MeCaman and Robins (5) developed a fluorometric test that quantitatively measures blood serum phenylalanine a t normal and abnormal levels. Another application of fluorometry to biomedical research is the study by Elevitch and Phillips of t,he lactate dehydrogenase isoenzymes (8). Damage to different organs ofthe body causes distinctive changes in the pattern of these isoenzymes which can therefore be useful for diagnosis. Fluorescence has also been used as a probe to study the structure of proteins. The native ultraviolet fluorescence of proteins was studied by Teale (4) in relation to the question of energy transfer in proteins and also in relation to the effect of solvent on the quantum yield of protein fluorescence. More recently
fluorescence studies have indicated that there is a conformational change ill the enzyme lysozyme when it interacts with substrates and competitive inhibitors (5). Fluorescence has been used to study the binding of small molecules ~r-hichfluoresce vhen they interact non-covalently with proteins. This phenomenon has been known for many years (6) and has recently been the subject of much investigation (7-19). There were t v o fluorescence experiments performed by the students. The first ir-as the determination of the fluorescence quantum yield of fluorescein. This experiment had the dual purpose of familiarizing the student with the calculation of quantum yield and introducing the necessary instrumentation. The second experiment deals vith the noncovalent interaction of a fluorescent probe with the enzyme wchymotrypsin. Experiment No. 1: Fluorescence Quantum Yield Theory
The fundamental equation for fluorescence (13-15) is that F
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lo(1 -
+
(1)
where F is the fluorescence intensity in quanta per unit time, I. is the incident intensity in quanta per unit time, ECD is the ahsorbance, and is the quantum yield. The two cases of interest are infinite dilution and infinite absorption. Case 1: By expansion of the term in the parentheses (16)
+
F 1 National Institutes of Health Postdoctoral Fellow. Present address: Chemistry Department, Western Washington State College, Bellingham, Washington 98225.
=
=
I d [2.3 ECD
- (-2.3 2ECD2) l -
If ECD < .O1 then F
lo2.3 ECD
+
(3)
so that for dilute solutions fluorescence is a linear function of concentration. Case 2: The other area of interest is that of infinite absorption where F=Ia+
(4)
From a knowledge of F and loit is possible to obtain the absolute quantum yield of a material. By using a standard of known quantum yield it is possible to calculate the quantum yield of unknown materials when unlmoum and known are in dilute solution. The unknown in this experiment is fluorescein and the standard is quinine sulfate. The equation needed to calculate the quantum yield of an unknown is the following 9"
=
Area., (1 ~rea,(1 - lo-"")
The subscripts u and s refer to unknown and standard, respect,ively, Area is the area enclosed between the emission spectrum and fluorescence blank spectrum, and A is the absorbance. For a discussion of this equation see Parker and Rees (14) and Turner (17,18). Fluorescence measurements were made with a Turner Model 210 Spectrofluorometer (17) which gives corrected emission spectra in units of relative quanta per unit bandwidth when used in either the fluorescence or luminescence mode of operation. It is, of course, necessary to use an instrument which gives emission spectra corrected for variation in monochromator efficiency with wavelength and for variation in the sensitivity of the light detector with wavelength. An alternate method is to use an uncorrected spectrofluorometer and apply the necessary correction factors. For a discussion of instrumentation see the articles by Lott (19) and Turner (17). Experimental
Approximately 5 mg of quinine sulfate dihydrste (Matheson, Coleman, and Bell) is dissolved in 100 ml of 0.1 IV HBOI. T h e absorbance of this solution is t,hen measured a t 366 mp. I t should he in the neighharhood of 0.4. If it is greater than 0.4 then it should be diluted to give an absorbance of 0.4 or less. I t is necossxy to know this absorbance precisely. A '/LO dilution of this material is then made and the diluted sample is used to record tho fluorescence spectrum. Approximately 2 mg of fluorescein (Eastman) is dissolved in 0.1 N NaON. The absorbance of this solution is then measured a t 366 mp. One makes the necessary dilutions in order to arrive a t an absorbance of 0.4 or less. Similarly, s. '/Mdilution is made and the diluted sample is used to report the fluorescence spectrum. The students each make up their own solutions and run their fluoreseerm spectre, on a Turner 210 Spectrofluorometer. The mode selector is used in the Fluortlscence mode, excitation wavelength is 366 mp, the excit,atian bandwidth is 10 mfi, emission bandwidth 2.5 m#, and the temperature in the cell is controlled a t 25°C by means of cireulabing water. The emission spectra of quinine sulfat,e and fluorescein are shown in Figures 1 and 2. I t is necessary to inkgrate bhe areas uaine a olanimtllor. Bv nsine the measured areas. measured dxorbanees, and quantum yield of quinine sulfate it is then possible to calculate the quantum yield for the unknown from eqn. (5). The student is referred to Melhuish (80) for the absolute quantum yield of quinine bisulfate. I t should he pointed out that Melhuish used quinine bisulfate and not quinine sulfate for this determination. However, this difference effects only the malar~~~
n
Figure 1. Fluorescence emision spectrum (relative quonta per unit bandwidth) of quinine rvlfote in 0.1 N H+O+ a t 25%
la
,k
1
) ,io
rn
rdo
ern
it1.#
Figure 2. Fluorescence emission spectrum (relative quanta per unit bandwidth) of fluorescein in 0.1 N NaOH at 25-C.
ity vdues (factor of 2 ) and will be inconsequential for dilute solutions. Also, i t should be born in mind that Melhuish experimentally determined the qnantum yield for 5 X lo-' M quinine bisulfate utilizing eqn. (4) for a. totally absorbing solution and found a value of 0.508. I t is necessary to correct this value far the effect of concent,ration as has been done by Melhuish (80). A decrease in the fluorescenco efficiency of a solute as the concentration is increased is due to the quenching of excited solute molecules by unexcited ones (self-quenching). If this queuching requires the close approach of the two solute molecules, then the change in the fluorescence efficiency with concentration should obey the Stern-Volmer law.
where & is the eflieiency a t iufinibe dilution, 6 is the efficiency a t the concentration C (moles/l), and K , is the self-quenching canstant. Using the K , value of 14.5 M-' one then determines the value of 40 = 0.546. The student should then verify that the concentration used in this experiment is close enough to infinite dilution so that 0.546 can he used for the standard. Results
The results for the quantum yield of fluorescein vere invariably low: 0.72,0.72,0.73, and 0.73. This is most probably due to impurities in the sample used. Literature values show quite a spread in the reported figures for the quantum yield of fluorescein. Some representative numbers are 0.85 by Parker and Rees (I/+),0.92 by Weber and Teale @I), 0.78 by Forster and Livingston (2&),0.84 by Vavilov ($$, 0.85 by Hellstrom ($41, and 0.85 by Umberger and La Aler (25). It would probably have been better to use 9-aminoacridine.HC1 as the unknown in this experiment. The quantum yield has been determined ( U ) , and this material would also serve to show the fine structure possible in fluorescence spectra. Experiment No. 2
I n 1954 Weber and Laurence (6) reported a class of compounds that has the interesting property of being non-fluorescent in aqueous solution but fluoresce when bound to protein. Two examples are as follows
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case of titration with fluorophore in the absence of modifier the equation that results is
It xas noted a t that time that solutions of these compounds alone in certain organic solvents are fluorescent. Also, aqueous solutions of the compounds become brightly fluorescent following the addition of adsorbing proteins such as serum albumin or heat denatured proteins. This phenomenon has been used to determine the concentration of plasma albumin fluorometrically (26), to study the interaction of related compounds with antibody (27, 38), to study the acid expansion of bovine serum albumin (89), and to study the thermally induced transition of L-chains of Bence-Jones proteins (SO). There is good evidence that TNS and ANS hind to hydrophobic sites on proteins. Hydrophobic bonding is one type of noncovalent force rhich is important to the maintenance of tertiary structure in proteins. This term mas first used by Rauzmann (31) to describe the tendency of nonpolar residues to avoid the aqueous phase and adhere to one another. Another viewpoint concerning the nature of hydrophobic interactions has been expressed by Klotz (33). For a recent review of this very important aspect of protein structure see the article by NCmethy (33). Stryer ($4) has studied the binding of ANS to apomyoglobin and apohemoglobin. The fluorescence quantum yield of ANS in water is 0.004 whereas in the ANS-apomyoglobin complex it reaches a value of 0.98. Furthermore, myoglobin does not hind the dye at all. By using a combination of fluorescence and equilibrium dialysis it was found that one mole of ANS is bound per mole of apomyoglobin. He also found that the ANS in the ANS-apomyoglobin complex could be expelled by adding heme vhich is evidence for ANS and heme binding to the same site. It has been shown by the X-ray work of Kendrew (36) that the heme group is in the interior of the molecule, surrounded almost entirely by nonpolar residues. McClure and Edelman (10) have investigated the mechanism of fluorescence of TNS. They found an increase in the fluorescence quantum yield of TNS with an increase in the number of methylene groups in pure alcohol solvents (no protein present). For instance they found the quantum yield to be essentially zero in water, 0.34 in methanol, 0.52 in ethanol, and 0.57 in npropanol. They also studied the quantum yield and related parameters of TRS fluorescence in aqueous solution with several proteins. This work was continued to the special case of the binding of TNS to a-chymotrypsin (11) and also the activation of chymotrypsinogen (12). Theory
McClure and Edelman (11) have presented a general mechanism which involves noncompetitive interaction between an enzyme E and two small molecules, a fluorophore TNS and a modifier of fluorescence PI. For the 376 / Journal of Chemical Educafion
where Kt is the dissociation constant for the formation of the binary complex E.TNS, I,,,. is the observed intensity of fluorescence after correction for selfabsorption, I.,,, represents the maximal fluorescence of ",*" the fluorophore-enzyme complex, and [TNS], is the total concentration of TNS. If one plots I,,,, versus I,,,,/ [TNSIt, the slope will be -Kr and the y intercept will be I I n deriving this expression the assumption is made %at I.,,,
= a, [E ,TNS]
(8)
where al is a proportionality constant relating the fluorescence intensity to the concentration of binary complex. Actually eqn. (7) is another form of the Scatchard equation (36, 57). This can be seen as follows: For identical and independent sites
where o ' [TNSIb/[Elt. The symbols [TNS], and [TNSIa refer to the concentrations of TNS in the free and bound state, respectively, [Elt is the total concentration of enzyme, Kbindis the intrinsic binding constant for the interaction between TNS and enzyme, and n is the number of sites on the protein. If one multiplies through by a [Elt and rearranges terms
also
By substituting eqn. (11) into eqn. (10) one finds
Expression (13) derived from the Scatchard equation differs from the expression of McClure and Edelman only in the use of [TNS], instead of [TNSIL. Experimental A 2 ml quantity of a dilute a-chymotrypsin solution ( A m = 3.13) a t pH 3 is pipebted into a euvette. Then 1 ml of a pH 7.8 buffer is added and the contents of the cuvette are mixed by placing a piece of parafilm over the top. The contents are allowed to equilibrate with respect to temperature in the spectrofluorometer and this baseline is recorded (no TNS present). Then 25 wl of a concentrated TNS solution is added and the contents of the cnvette are mixed. The concentration of the stock M, and the solution of TNS is in the neighborhood of 3 X exact concentration can he calculated from the absorbance a t 366 mp. After equilibration the fluorescence emission spectrum is recorded. This procedure is repeated until 5 additions of TNS have been made. The mode selector is used in the Luminescence mode, and the other instrument settings are t,he same as in the first experiment. I t is necessary to measure the areas under the fluorescence curves using a planimeter, i.e., the net areas between the fluares-
cenee curves and baseline. The concentration of TNS in the cuvette is calculated from the absorbance a t 366 mp using the molar absorptivity 4100 M-' cm-1 (10) and taking into accmlnt the dilation of the TNS. The dilution of the protein by buffer is considered but the dilution of the protein hy added TNS is ignored. It is a well-known fact that fluorescence measurements are linear with concentration only in very dilute solution (See the Theory section of the first experiment). It is therefore necessary to correct for this effect if one assumes a linear relation to exist. The measured integrated intensities are corrected for selfabsorption using the equation of McClure and Edelman (11)
where [TNS]t is the total TNS concentration, is the molar absorptivity of TNS a t the exciting wavelength, and I,.,, and Ioba refer to the corrected and observed intensities, respectively. This equation will apply to an experimental situation where one uses a. 1 centimeter square euvette, and the fluorescence is detected a t right angles to the incident light.
Results
The titration of a-chymotrypsin with TIVS is shown in Pigure 3. The areas of the fluorescence curves are inteerated and these values are corrected for selfabsorption using eqn. (14). A plot of I,,, versus I,,,./
-
useful comments on the contents of the manuscript. This investigation was supported (in part) by a U.S. Public Health Service fellomhip (2F2 A31 17, 231-02) from the Institute of Arthritis and Metabolic Diseases, the U.S. Public Health Service. Literature Cited (1) Chemical and Engineering News, 45, 72 (1967). (2) PHILLIPS, R. E., A N D ELEVITCH, F. R., "Progress in Clinical Patholoev." Vol. 1. Grme and Stratton. New York, 1966,
Chapte;2: (3) MCCAMAN, M. W., AND ROBINB,E., J . Lab. and Clin. Med., 59, 885 (1962). (4) T e n m , F. W. J., Biochem. J., 76, 381 (1960).
(5) LEHRER,S. S., AND FASMAN, G. D., Biochem. Biophgs. Res. Comm., 23, 133 (1966). (61 D. J. R., Bioehem. J.,56, xxxi . . WEBER,G., A N D LAURENCIS,
(1954). (7) DANIEL, E., A N D W m m , G., Biochemistry, 5, 1893 (1966). (8) WZBISR,G., A N D DANIISL, E., Biochemistry, 5, 1900 (1966). (9) ALEXANDER, B. A,, A N D EDI:LMAN, G. M., Fed. Proc., 24,413 (1965). (10) MCCLURE,W. O., A N D EDELMAN, G. If., Bioehemislry, 5, 1908 (1966). (11) G. hl., Biochemistry, 6, . . MCCLURE,W. O., A N D EDELMAN, 559 (1967). (12) W. 0.. A N D EDELMAN. G. M., Biochemisln~,6, , , MCCLURI:. . 567 (1967). (13) WHITE:,C. E., "Trace Analysis," John Wiley & Sons, Inc., New York, 1957. (14) PARKER, C. A,, A N D Rt:~:s, W. T., Analgst, 85, 587 (1960). D. hl., Anal. Chern., 38, 29A (1966). (15) HERCULES, 116) BAUNSBERG. H.. AND OSBORN. S. B.. Anal. Chim. Ada., 6, , 84 (1952): ' (17) TURNER, G. K., Science, 146, 183 (1964). (18) TURNER, G. K., Operating Manual for Model 210 Spectro-
.
Figure 3. Fluorexence titmtion a-chymotryptin with TNS. 25 p l additions of TNS (3.41 X 10-3 MI to 3.0 ml of a-chymotryprin 14.05 X MI at pH of
7.8 and 25'C.
[TNS], according to eqn. (7) is shown in Figure 4 and K , is determined from the slope. The values obtained for Kr by the 4 students doing this experiment were as follows: 1.9 X M, 2.2 X M, 2.7 X 10-4 A t , and 4.2 X M . The literature value (11) is given as 2.05 X 10W4M. Acknowledgments
I am indebted to Mr. Peter Grogg for the preparation of TNS. I would like to thank Dr. W. 0. IIcClure and Dr. G. M. Edelman for providing a sample of TNS for purposes of comparison and for reprints of their papers before publication. The author is indebted to Dr. Hans Neurath for his encouragement and support of this project. Thanks are due to Mr. Ralph Kenner for doing a "dry-run" of these experiments. The author would also like to thank Mr. Ralph Kenner, Mr. George Turner, and Dr. David Teller for their
fluommeter, G. K . Turner Associates, P d o Alto, Cdifornia. (19) LOW, P. F., J. CHEM.EDUC., 41, A327 and A421 (1964). (20) MELHUISH, W. II., J. Phys. Chem., 65, 229 (1961). 1211 W m m . G.. A N D TEAL],:.F. W. J.. Trans. Faradau Soc., 53, ,
.
646 (i957). (22) FORSTER, L. S., A N D LIVINGSTON, R., J . Chem. Phys., 20, 1315 (1952). (23) VAVIWV,S. I., Z. P h y ~ i k22, , 266 (1924). (24) HELLSTROM, IT., Arkiu. Kemi. hfin. Geol., 12A, 17 (1937). (25) UMBJ:ROE~, J., A N D LAMER,V., J . Am. Chem. Soc., 67, 1099 (1945). (261 Fnm:s. J. E.. LAURENCI:. D. J. R., RI:ES, V. H., Biochem. , J., 56, xxxi (1954). (27) WINKLER, M., J . Mol. B i d , 4, 118 (1962). (28) B E R N ~D. , S., AND SrNGlm, S. J., Immunochemistry, 1, 209 (1964). (29) Wli.nm, G., A N D YOUNG,I,. B., J. Biol. Chem., 239, 1415 (1964). (30) GALLY, J. A,, ANDEDELMAN, G. M., Biochem. Biophys. Ada, 94, 175 (1965). (31) KAUZMANN,W., "The Mechanism of Enzyme Action," The John Hopkins Press, Baltimore, 1954, p. 71. (32) KWTZ, I. W., Brookhaven Symposia, in Biology, 13, 25 (1960). (33) N B M ~ K Y G.,, Angew. Chem. Internal. Edit., 6, 195 (1967). (34) ~ T R Y I ~ RL., , J . Mol. Biol., 13, 482 (1965). (35) J. C., Brookhaven Symposi& in Biology, 15, 216 . . . KI:NDREW. (1962). (36) SCATCHARD, G., Annals o j N.Y. Aead. Sci., 51, 660 (1949). (37) KLOTZ,I. M., "The Proteins" (1st Ed.) Academic Press, Inc., New York, 1953, Vol. 13, p. 727.
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