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The rotational relaxation time of aspartate aminotransferase
BIOCHIMICA ET BIOPHYSICA ACTA
5II
BBA 35134 T H E ROTATIONAL R E L A X A T I O N T I M E OF ASPARTATE AMINOTRANSFERASE J O R G E E. CHURCHICH
Department of Biochemistry, University of Tennessee, Knoxville, Tenn. (U.S.A.) (Received June i6th, 1967)
SUMMARY
Two independent methods, sucrose density centrifugation and polarization of fluorescence, were used to study the molecular state of the enzyme aspaltate aminotransferase at concentrations approaching those used in the enzymatic assays. I. The sucrose density centrifugation experiments indicated that the sedimentation coefficient of the enzyme (5.5 S) is not appreciably affected by the simple dilution process. 2. The polarization of fluorescence results, conducted at a protein concentration of o.oi mg/ml, revealed that the enzyme aspartate aminotransferase is characterized by two rotational relaxation times. The longest relaxation time (ph = 13o nsec) corresponds to a rigid macromolecule unit of a mol. wt. of approx. 9oooo. The shortest relaxation time ( p s - - 9 nsec) reflects the Brownian motion of a short segment of the enzyme.
INTRODUCTION
The enzyme aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase EC 2.6.I.I) contains two molecules of pyridoxal 5-phosphate per mol. wt. of u o o o o (ref. I) and a quantitative determination of the terminal alanine residues showed that there are two molecules of alanine per mole of protein s. Although these results appear to indicate that the enzyme is composed of two polypeptide chains, the molecular weight measurements, normally conducted at protein concentrations of the order of 5 mg/ml, did not reveal the presence of species of lower molecular weight 3& Since the two polypeptide chains m a y have a tendency to associate at high protein concentrations, it was thought of interest to investigate the molecular state of the enzyme at concentrations approaching those used in the enzymatic assays. To this end the method of polarization of fluorescence was used to determine the rotational relaxation time of the enzyme at a concentration of o.oi mg/ml. METHODS
Polarization of fluorescence measurements Polarization of fluorescence measurements were performed in a photometer built in our laboratory. Illumination was provided by a Xenon lamp (Hanovia Biochim. Biophys. Acta, 147 (1967) 511-517
512
J. E. CKURCHICH
15o W) with wavelengths selected by a quartz prisnl monochromator (Schoeffel, QPM, 3oS). Light incident along the excitation axis was polarized with a Glan Thompson prism (I2,I2 nnn aperture, Crystal Optics, Chicago). An identical arrangement mounted at a right angle to the excitation beam was used to select polarized light along V and H, vertical and horizontal axes, respectively. The emitted light passed through the filter combination 2 M NaNO2 and Corning CS 3-7 o. A photomultiplier (EMI 95o2 B) attached to an electrometer (Keitley, 61o B) was used as detector. This arrangement gave typical signals of 3" IO-6 A, with photomultiplier dark current less than I . I O - u A. An analysis of the various sources of random and systematic errors shows that this apparatus is capable of measuring degree of polarization of fluorescence values to an accuracy of I o/ ,.o for polarization values greater than o.I. Fluorescence spectra were recorded in a spectrofluorinleter equipped with two Bausch and Lomb monochromators. The slits were set to give a band width of 3 m/~. Fluorescence quantum yields of the dye coupled to the enzyme were determined by comparing the total emission from the dye-protein conjugates with that of standard dye solutions. The absolute quantum yield of fluorescein isothiocyanate was taken as o.65 (ref. 5) and the lifetime of the excited state r ~ 5" IO 9 sec (ref. 6). Sucrose demity centrifugatio~ The procedure described by MARTIN AND AMES7 was used. The gradients were made from 2. 3 ml each of 5 and 2o % sucrose solutions made in o.I M phosphate buffer (pH 7.2). Beef-liver catalase (Worthington), egg white lysozyme (Sigma), horse alcohol dehydrogenase (Sigma) and yeast alcohol dehydrogenase (Sigma) were used as standards. A solution (o.I ml) containing catalase (o.o5 rag), lysozyme (o.25 rag), alcohol dehydrogenase (o. I rag) and aspartate aminotransferase (o.I mg) was carefully layered on the gradients. The samples were centrifuged in the rotor SW-39 in the Spinco Model L ultracentrifuge at 38ooo rev./min for 14 h at 4 °. The samples were fractionated by puncturing the bottom of the tubes and collecting samples of approx. o.I5 ml each. The fractions were assayed for enzymatic activity according to the procedures described MARTIN AND A.MES7. The activity of aspartate transaminase was assayed according to the method of SrzEI~ AND JExI
Biochim. Biophys. Acta, I47 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
513
RESULTS AND DISCUSSION
Sucrose densifv-gradient centrifugation The effect of binding of fluorescein isothiocyanate on the sedimentation properties of aspartate aminotransferase was examined by sucrose density centrifugation. The activity distribution curves are given in Fig. I and the sedimentation values are summarized in Table I. As shown in Fig. i, the tubes exhibiting aminotransferase activity had identical distribution patterns, indicating that differences, if any, in size and shape between the native and labelled species are relatively small. In addition, the sedimentation values included in Table I agree closely with those obtained in the analytical ultracentrifuge at larger protein concentrations3, 4. In the context of the present experiments, it seems reasonable to propose that the hydrodynamic properties of the enzyme aspartate aminotransferase are unaffected by binding of fluorescein isothiocyanate.
Fhtorescence spectra The fluorescence properties of free and bound fluorescein isothiocyanate were studied in phosphate buffer over the pHi range from 6.8 to 7.5. Fig. 2 shows the corTABLE I SEDIMENTATION COEFFICIENTS DETERMINED FROM SUCROSE DENSITY-GRADIENT CENTRIFUGATION T h e following Seo,w v a l u e s were u s e d for calculation: catalase 11.3 (ref. 7), m u r a m i d a s e 1.91 (ref. I4), horse alcohol d e h y d r o g e n a s e 5.1 (ref. 15), y e a s t alcohol d e h y d r o g e n a s e 7.2 (ref. 16).
Standard
Aspartate aminolransferase
Catalase Muramidase H u m a n alcohol d e h y d r o g e n a s e Y e a s t alcohol d e h y d r o g e n a s e
z
0%
<
!
Native
Labelled
5.6 5.I 5.8 5.6
5.5 5.2 5.8 5.5
o~
~ ~u
005
0
5
Io
15
DISTANCE
20 ~ROM
25
30
MENISCUS (CM~
35
Fig. I. E n z y m a t i c a c t i v i t y d i s t r i b u t i o n in a sucrose d e n s i t y gradient. S t a n d a r d s n l u r a m i d a s e (O), y e a s t alcohol d e h y d r o g e n a s e (<_~), horse alcohol d e h y d r o g e n a s e ( y ) , catalase ([3). T o p g r a p h : E n z y m a t i c a c t i v i t y d i s t r i b u t i o n of fluorescein i s o t h i o c y a n a t e - a m i n o t r a n s f e r a s e ( 0 ) a n d fluorescence i n t e n s i t y m e a s u r e m e n t s a t 5zo m p (excitation 48o m/t ) (V). B o t t o m g r a p h : e n z y m a t i c a c t i v i t y d i s t r i b u t i o n of a m i n o t r a n s f e r a s e ( 0 ) .
Biochim. Biophys. Acta, 147 (1967) 511-517
514
]. E. CHURCHICH
r e c t e d e m i s s i o n s p e c t r a o b t a i n e d b y e x c i t a t i o n w i t h l i g h t of 480 m/x. I t is i m m e d i a t e l y a p p a r e n t t h a t t h e p o s i t i o n of t h e e m i s s i o n b a n d is n o t a f f e c t e d w h e n t h e f l u o r o p h o r is a t t a c h e d to t h e e n z y m e . Tile p o l a r i z a t i o n of fluorescence spectra, w h i c h is t h e set of v a l u e s of t h e p o l a r i z a t i o n r e c o r d e d u p o n e x c i t a t i o n w i t h l i g h t of v a r y i n g w a v e l e n g t h , is r e p r e s e n t e d in Fig. 2. T h e p o l a r i z a t i o n s p e c t r m n of b o u n d fluorescein shows a w a v e l e n g t h - i n d e p e n d e n c e of t h e e x c i t i n g l i g h t t h r o u g h o u t t h e m a i n a b s o r p t i o n b a n d ( 4 4 o - 4 8 o m/x). H o w e v e r , t h e a b s o l u t e v a l u e of t h e p o l a r i z a t i o n of fluorescence (P 0.29) is s i g n i f i c a n t l y s m a l l e r t h a n t h e v a l u e e x p e c t e d for a f l u o r o p h o r in a r i g i d m e d i u m s u c h as g l y c e r o l (P o.42 ).
0 52 . . . .
°%22:
4O 50 2o
o2)
10 4~o
°l I 520 550 570 ), (M~]
440 460 480 x
(~}x]
Fig. 2. Left: Fluorescence spectra of isothiocyanate fluorescein (O) and fluorescein isothiocyanateaminotransferase (O) in o.i M phosphate buffer (pH %5). Fluorescein isothiocyanate-aminotransferase in 60°/0 sucrose (V). Temperature of the samples 25 °. Right: Polarization of fluorescence spectra of fluorescein isothiocyanate in 95 % glycerol (O), fluorescein isothiocyanateaminotransferase in o.i M phosphate buffer (pH 7.5) (O), fluorescein isothiocyanate-aminotransferase in 60 o/0 sucrose (V). Temperature of the system 25 °. TABLE II R O T A T I O N A L R E L A X A T I O N T I M E S OF F L U O R E S C E I N I S O T t l l O C Y A N A T E - - A S P A R T A T E A M I N O T R A N S F E R A S E
p, apparent rotational relaxation time; Po, limiting polarization of fluorescence; q, quantum yield of fluorescence; ph, rotational relaxation time (harmonic mean) of the protein; ps, rotational relaxation time of the "side chain". A. T]~1 was changed by increasing or decreasing the temperature. Protein conch, (mg/ml)
pH
p
Po
q
o.oi o.i 0.3 O.Ol o.i
6.8 6.8 6.8 7.5 7.5
80 85 82 81 84
0.34 0.34 o.34 0.34 0-34
0.62 0.62 o.63 o.61 0.62
B. 7"/~1 was varied by addition of sucrose. Measurements conducted at 25 °. Protein cohen,
pH
ph
ps
Po
q
6.8 7.5 6.8
13o 13o 135
9.0 9.2 9.4
0.38 0.38 0.38
0.60 o.61 0.62
(rag/m/) o.o~ o.o~ 0. 3
Bioehim. Biophys. Acta, 147 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
515
A behavior of this kind is expected since it is well established that the Brownian rotation of either the macromolecule or the fluorophor would tend to diminish the polarization of fluorescence of the system. A quantitative study of the fluorescence quantum yield values of fluorescein isothiocyanate attached to the protein is summarized in Table II. It is evident that the quantum yield of bound fluorescein is not appreciably different from that of the free dye. In addition, the q values are insensitive to variations in the pH of the medium, at least within the pH region examined. The fact that the spectroscopy properties of bound fluorescein are similar to that exhibited by tile free dye suggests that the environment of the protein has no effect on the lifetime of the excited state. This assumption was introduced in the analysis of the rotational relaxation times as determined by polarization of fluorescence.
Delermb~atio~ of the rotational relaxatio~ time Fig. 3 shows a plot of I / f l - - 1 / 3 vs. T/r] for the dye-protein conjugates at a protein concentration of approx, o.oi mg/ml. The plot is linear within experimental error for the range 5-35 ° and fits Perrin's equation (Eqn. I). I P
I 3
-
(~00
--
~) ( 3I ~+h ~ )
(i)
In tile above equation P0 is the limiting polarization of fluorescence, z, lifetime of the excited state and ph, the relaxation time (harmonic mean).
-la-25i I ~/
2
0
l;O
Ya
2;0
5JO0
] 400
Fig. 3. Polarization of fluorescence results for fluorescein isothiocyanate-aminotransferase at a protein concentration of o.oi mg/ml. Results obtained at pH 7.5 (Y) and pH 6.8 (V) when T/rI was changed by increasing or decreasing the temperature of the solution. Results obtained with fluorescein isothiocyanate-aminotransferase at pH 6.8 (O) and pH 7.5 (0) in sucrose solutions at 25 °. The wavelength of excitation was 48o m M.
Taking the lifetime of the excited state to be r = 5" lO-9 sec the apparent relaxation time at 25 ° was found to be 8o nsec (Table II). Similar results were obtained when the relaxation times were determined at protein concentrations of o.I and o. 3 mg/ml. The analysis of the polarization results according to Eqn. I did not take into account the contribution of the rotational freedom of the dye to the calculated relaxation time. If the fluorophor bound to the enzyme has some degree of internal rotation then it might be anticipated that the calculated relaxation time should be smaller than the value expected for the rotation of the entire macromolecule in soBiochim. Biophys. Acta, 147 (1967) 5II-5IZ
510
J . E . CHURCHICH
lution. In an attempt to evaluate tile relaxation time of tile enzyme, the technique proposed by GOTTBIEB AXD \VAHLn was applied to the polarization measurements. Accordingly, the parameter P was determined at constant temperature (25 °) and the viscosity of the medium was changed by addition of sucrose. Under this new set of experimental conditions, the plot of I / P - - 1 / 3 vs. T/r 1 shows a downward curvature at low T/r~ values. This type of curvature m a y occur whenever more than one relaxation time is present. Following the theoretical treatment proposed by MEMMINGr2 a n d GOTTBIEB AND \*rAHLll, it is possible to analyze the composite depolarization of fluorescence due to the rotation of the macromolecule (ph) and to the fast rotation of a short segment of the protein to which the dye is loosely bound (ps). If Oh > ps then Eqn. 2 can be applied to the analysis of the polarization data.
1 1 (1 -
p0-o
1'() . -
1
') (-'!
,
In this equation the parameter/3 is a function of the angles 81, 82 between the axis of rotation and the oscillators of emission and absorption, respectively. The rotational relaxation time ph and ps are related to the temperature and viscosity of the medium: 3v
K1T
3v
K2T
Oh
tj
ps
~]
(3)
Eqn. 2 adequately represents the experimental results obtained in sucrose solutions provided the parameters K 1 , K 2 and fl have the following values" K t 3.7" IO-6, K2 ~ 5" IO-5 and fl = o.8. The calculated relaxation times are included in Table II. The most salient feature of these results is that the pH of the medium has little effect on both ph and ps. Furthermore, the relaxation times pla and ps differ by more than Io-fold and this exact difference was reproducible in a number of different measurements. The shortest relaxation time m a y be tentatively assigned to a rotating unit of low molecular weight, presumably a segment of tile protein (mol. wt. 5ooo) to which the fluorophor is attached. The longest relaxation time, on the other hand, reflects the rotation of the enzyme in solution. A relaxation time of this order of magnitude (13o nsec) is in conformity with that expected for all ellipsoid of approx, mol. wt. 9oooo (axial ratio 4) la. Since a molecular weight of this order of magnitude (IOOooo) has been previously determined by equilibrium ultracentrifugation s, it is concluded that the quaternary structure of the protein is not affected at concentrations approaching those used in the enzymatic assays. ACKNOWLEDGEMENTS
The author wishes to thank Dr. T. SALO for helpful advice in the sucrose density gradient experiments. The technical assistance of M. BATTISTAis acknowledged. This research was supported by a grant from the Atomic Energy Commission (AT 4o1-3553). Biochim. Biophys. Acta, 147 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
517
REFERENCES i R. C. HUGHES, W. T. JENKINS AND E. H. FISHER, Proc. Natl. Acad. Sei. U.S., 48 (1962) 16I 5. 2 C. TURANO, A. GUARTOSlO, F. RlVA AND P. VECCHINI, Chem. Biol. Aspects Pyridoxat Catalysis, Proc. Syrup. 1.U.B. Rome, I962, P e r g a m o n Press, London, 1965, p- 149. 3 ~,V. T. JENKINS, D. A. YPHANTIS AND J. ~vV. SIZER, jr. Biol. Chem., 234 (1959) 51. 4 J. E. CHURCHlCH AND L. HARPRING, Biochim. Biophys. Acta, lO 5 (1965) 5755 G. WEBER AND F. W. TEALE, Trans. Faraday Soc., 53 (1967) 646. 6 R. F. STEINER AND A. J. McLISTER, J. Polymer Sci., 24 (1957) lO5. 7 R. G. MARTIN AND B. N. AMES, J. Biol. Chem., 236 (196o) 1372. 8 J. W. SIZBR AND W. T. JENKINS, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, VoI. 5, Academic Press, New York, 1962, p. 677. 9 J. E. CHURCHICH, Bioehim. Biophys. Aeta, 65 (1962) 349. IO O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . I I Y. GOTTBIEB AND P. WAHL, J. Chim. Phys., 6o (1963) 849. 12 R. MEMMING, Z. Physiol. Chem., 28 (1961) 168. 13 G. WEBER, Biochem. J., 51 (1952) 145. 14 A. J. SOPHIANOPOULOS, C. K. RHODES, C. N. HOLCOMB AND K. E. VAN HOLOE, J. Biol. Chem., 237 (1962) 11o 7 . 15 K. DALZlEI. AND A. EHRENBERG, Acla Chem. Scan&, 12 (1958) 465 . 16 E. L. KUFF, G. H. HOGEBOOM, M. J. STRIEBICH, jr. Biol. Chem., 212 (1955) 439.
Biochim. Biophys. Acta, 147 (1967) 511-517
5II
BBA 35134 T H E ROTATIONAL R E L A X A T I O N T I M E OF ASPARTATE AMINOTRANSFERASE J O R G E E. CHURCHICH
Department of Biochemistry, University of Tennessee, Knoxville, Tenn. (U.S.A.) (Received June i6th, 1967)
SUMMARY
Two independent methods, sucrose density centrifugation and polarization of fluorescence, were used to study the molecular state of the enzyme aspaltate aminotransferase at concentrations approaching those used in the enzymatic assays. I. The sucrose density centrifugation experiments indicated that the sedimentation coefficient of the enzyme (5.5 S) is not appreciably affected by the simple dilution process. 2. The polarization of fluorescence results, conducted at a protein concentration of o.oi mg/ml, revealed that the enzyme aspartate aminotransferase is characterized by two rotational relaxation times. The longest relaxation time (ph = 13o nsec) corresponds to a rigid macromolecule unit of a mol. wt. of approx. 9oooo. The shortest relaxation time ( p s - - 9 nsec) reflects the Brownian motion of a short segment of the enzyme.
INTRODUCTION
The enzyme aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase EC 2.6.I.I) contains two molecules of pyridoxal 5-phosphate per mol. wt. of u o o o o (ref. I) and a quantitative determination of the terminal alanine residues showed that there are two molecules of alanine per mole of protein s. Although these results appear to indicate that the enzyme is composed of two polypeptide chains, the molecular weight measurements, normally conducted at protein concentrations of the order of 5 mg/ml, did not reveal the presence of species of lower molecular weight 3& Since the two polypeptide chains m a y have a tendency to associate at high protein concentrations, it was thought of interest to investigate the molecular state of the enzyme at concentrations approaching those used in the enzymatic assays. To this end the method of polarization of fluorescence was used to determine the rotational relaxation time of the enzyme at a concentration of o.oi mg/ml. METHODS
Polarization of fluorescence measurements Polarization of fluorescence measurements were performed in a photometer built in our laboratory. Illumination was provided by a Xenon lamp (Hanovia Biochim. Biophys. Acta, 147 (1967) 511-517
512
J. E. CKURCHICH
15o W) with wavelengths selected by a quartz prisnl monochromator (Schoeffel, QPM, 3oS). Light incident along the excitation axis was polarized with a Glan Thompson prism (I2,I2 nnn aperture, Crystal Optics, Chicago). An identical arrangement mounted at a right angle to the excitation beam was used to select polarized light along V and H, vertical and horizontal axes, respectively. The emitted light passed through the filter combination 2 M NaNO2 and Corning CS 3-7 o. A photomultiplier (EMI 95o2 B) attached to an electrometer (Keitley, 61o B) was used as detector. This arrangement gave typical signals of 3" IO-6 A, with photomultiplier dark current less than I . I O - u A. An analysis of the various sources of random and systematic errors shows that this apparatus is capable of measuring degree of polarization of fluorescence values to an accuracy of I o/ ,.o for polarization values greater than o.I. Fluorescence spectra were recorded in a spectrofluorinleter equipped with two Bausch and Lomb monochromators. The slits were set to give a band width of 3 m/~. Fluorescence quantum yields of the dye coupled to the enzyme were determined by comparing the total emission from the dye-protein conjugates with that of standard dye solutions. The absolute quantum yield of fluorescein isothiocyanate was taken as o.65 (ref. 5) and the lifetime of the excited state r ~ 5" IO 9 sec (ref. 6). Sucrose demity centrifugatio~ The procedure described by MARTIN AND AMES7 was used. The gradients were made from 2. 3 ml each of 5 and 2o % sucrose solutions made in o.I M phosphate buffer (pH 7.2). Beef-liver catalase (Worthington), egg white lysozyme (Sigma), horse alcohol dehydrogenase (Sigma) and yeast alcohol dehydrogenase (Sigma) were used as standards. A solution (o.I ml) containing catalase (o.o5 rag), lysozyme (o.25 rag), alcohol dehydrogenase (o. I rag) and aspartate aminotransferase (o.I mg) was carefully layered on the gradients. The samples were centrifuged in the rotor SW-39 in the Spinco Model L ultracentrifuge at 38ooo rev./min for 14 h at 4 °. The samples were fractionated by puncturing the bottom of the tubes and collecting samples of approx. o.I5 ml each. The fractions were assayed for enzymatic activity according to the procedures described MARTIN AND A.MES7. The activity of aspartate transaminase was assayed according to the method of SrzEI~ AND JExI
Biochim. Biophys. Acta, I47 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
513
RESULTS AND DISCUSSION
Sucrose densifv-gradient centrifugation The effect of binding of fluorescein isothiocyanate on the sedimentation properties of aspartate aminotransferase was examined by sucrose density centrifugation. The activity distribution curves are given in Fig. I and the sedimentation values are summarized in Table I. As shown in Fig. i, the tubes exhibiting aminotransferase activity had identical distribution patterns, indicating that differences, if any, in size and shape between the native and labelled species are relatively small. In addition, the sedimentation values included in Table I agree closely with those obtained in the analytical ultracentrifuge at larger protein concentrations3, 4. In the context of the present experiments, it seems reasonable to propose that the hydrodynamic properties of the enzyme aspartate aminotransferase are unaffected by binding of fluorescein isothiocyanate.
Fhtorescence spectra The fluorescence properties of free and bound fluorescein isothiocyanate were studied in phosphate buffer over the pHi range from 6.8 to 7.5. Fig. 2 shows the corTABLE I SEDIMENTATION COEFFICIENTS DETERMINED FROM SUCROSE DENSITY-GRADIENT CENTRIFUGATION T h e following Seo,w v a l u e s were u s e d for calculation: catalase 11.3 (ref. 7), m u r a m i d a s e 1.91 (ref. I4), horse alcohol d e h y d r o g e n a s e 5.1 (ref. 15), y e a s t alcohol d e h y d r o g e n a s e 7.2 (ref. 16).
Standard
Aspartate aminolransferase
Catalase Muramidase H u m a n alcohol d e h y d r o g e n a s e Y e a s t alcohol d e h y d r o g e n a s e
z
0%
<
!
Native
Labelled
5.6 5.I 5.8 5.6
5.5 5.2 5.8 5.5
o~
~ ~u
005
0
5
Io
15
DISTANCE
20 ~ROM
25
30
MENISCUS (CM~
35
Fig. I. E n z y m a t i c a c t i v i t y d i s t r i b u t i o n in a sucrose d e n s i t y gradient. S t a n d a r d s n l u r a m i d a s e (O), y e a s t alcohol d e h y d r o g e n a s e (<_~), horse alcohol d e h y d r o g e n a s e ( y ) , catalase ([3). T o p g r a p h : E n z y m a t i c a c t i v i t y d i s t r i b u t i o n of fluorescein i s o t h i o c y a n a t e - a m i n o t r a n s f e r a s e ( 0 ) a n d fluorescence i n t e n s i t y m e a s u r e m e n t s a t 5zo m p (excitation 48o m/t ) (V). B o t t o m g r a p h : e n z y m a t i c a c t i v i t y d i s t r i b u t i o n of a m i n o t r a n s f e r a s e ( 0 ) .
Biochim. Biophys. Acta, 147 (1967) 511-517
514
]. E. CHURCHICH
r e c t e d e m i s s i o n s p e c t r a o b t a i n e d b y e x c i t a t i o n w i t h l i g h t of 480 m/x. I t is i m m e d i a t e l y a p p a r e n t t h a t t h e p o s i t i o n of t h e e m i s s i o n b a n d is n o t a f f e c t e d w h e n t h e f l u o r o p h o r is a t t a c h e d to t h e e n z y m e . Tile p o l a r i z a t i o n of fluorescence spectra, w h i c h is t h e set of v a l u e s of t h e p o l a r i z a t i o n r e c o r d e d u p o n e x c i t a t i o n w i t h l i g h t of v a r y i n g w a v e l e n g t h , is r e p r e s e n t e d in Fig. 2. T h e p o l a r i z a t i o n s p e c t r m n of b o u n d fluorescein shows a w a v e l e n g t h - i n d e p e n d e n c e of t h e e x c i t i n g l i g h t t h r o u g h o u t t h e m a i n a b s o r p t i o n b a n d ( 4 4 o - 4 8 o m/x). H o w e v e r , t h e a b s o l u t e v a l u e of t h e p o l a r i z a t i o n of fluorescence (P 0.29) is s i g n i f i c a n t l y s m a l l e r t h a n t h e v a l u e e x p e c t e d for a f l u o r o p h o r in a r i g i d m e d i u m s u c h as g l y c e r o l (P o.42 ).
0 52 . . . .
°%22:
4O 50 2o
o2)
10 4~o
°l I 520 550 570 ), (M~]
440 460 480 x
(~}x]
Fig. 2. Left: Fluorescence spectra of isothiocyanate fluorescein (O) and fluorescein isothiocyanateaminotransferase (O) in o.i M phosphate buffer (pH %5). Fluorescein isothiocyanate-aminotransferase in 60°/0 sucrose (V). Temperature of the samples 25 °. Right: Polarization of fluorescence spectra of fluorescein isothiocyanate in 95 % glycerol (O), fluorescein isothiocyanateaminotransferase in o.i M phosphate buffer (pH 7.5) (O), fluorescein isothiocyanate-aminotransferase in 60 o/0 sucrose (V). Temperature of the system 25 °. TABLE II R O T A T I O N A L R E L A X A T I O N T I M E S OF F L U O R E S C E I N I S O T t l l O C Y A N A T E - - A S P A R T A T E A M I N O T R A N S F E R A S E
p, apparent rotational relaxation time; Po, limiting polarization of fluorescence; q, quantum yield of fluorescence; ph, rotational relaxation time (harmonic mean) of the protein; ps, rotational relaxation time of the "side chain". A. T]~1 was changed by increasing or decreasing the temperature. Protein conch, (mg/ml)
pH
p
Po
q
o.oi o.i 0.3 O.Ol o.i
6.8 6.8 6.8 7.5 7.5
80 85 82 81 84
0.34 0.34 o.34 0.34 0-34
0.62 0.62 o.63 o.61 0.62
B. 7"/~1 was varied by addition of sucrose. Measurements conducted at 25 °. Protein cohen,
pH
ph
ps
Po
q
6.8 7.5 6.8
13o 13o 135
9.0 9.2 9.4
0.38 0.38 0.38
0.60 o.61 0.62
(rag/m/) o.o~ o.o~ 0. 3
Bioehim. Biophys. Acta, 147 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
515
A behavior of this kind is expected since it is well established that the Brownian rotation of either the macromolecule or the fluorophor would tend to diminish the polarization of fluorescence of the system. A quantitative study of the fluorescence quantum yield values of fluorescein isothiocyanate attached to the protein is summarized in Table II. It is evident that the quantum yield of bound fluorescein is not appreciably different from that of the free dye. In addition, the q values are insensitive to variations in the pH of the medium, at least within the pH region examined. The fact that the spectroscopy properties of bound fluorescein are similar to that exhibited by tile free dye suggests that the environment of the protein has no effect on the lifetime of the excited state. This assumption was introduced in the analysis of the rotational relaxation times as determined by polarization of fluorescence.
Delermb~atio~ of the rotational relaxatio~ time Fig. 3 shows a plot of I / f l - - 1 / 3 vs. T/r] for the dye-protein conjugates at a protein concentration of approx, o.oi mg/ml. The plot is linear within experimental error for the range 5-35 ° and fits Perrin's equation (Eqn. I). I P
I 3
-
(~00
--
~) ( 3I ~+h ~ )
(i)
In tile above equation P0 is the limiting polarization of fluorescence, z, lifetime of the excited state and ph, the relaxation time (harmonic mean).
-la-25i I ~/
2
0
l;O
Ya
2;0
5JO0
] 400
Fig. 3. Polarization of fluorescence results for fluorescein isothiocyanate-aminotransferase at a protein concentration of o.oi mg/ml. Results obtained at pH 7.5 (Y) and pH 6.8 (V) when T/rI was changed by increasing or decreasing the temperature of the solution. Results obtained with fluorescein isothiocyanate-aminotransferase at pH 6.8 (O) and pH 7.5 (0) in sucrose solutions at 25 °. The wavelength of excitation was 48o m M.
Taking the lifetime of the excited state to be r = 5" lO-9 sec the apparent relaxation time at 25 ° was found to be 8o nsec (Table II). Similar results were obtained when the relaxation times were determined at protein concentrations of o.I and o. 3 mg/ml. The analysis of the polarization results according to Eqn. I did not take into account the contribution of the rotational freedom of the dye to the calculated relaxation time. If the fluorophor bound to the enzyme has some degree of internal rotation then it might be anticipated that the calculated relaxation time should be smaller than the value expected for the rotation of the entire macromolecule in soBiochim. Biophys. Acta, 147 (1967) 5II-5IZ
510
J . E . CHURCHICH
lution. In an attempt to evaluate tile relaxation time of tile enzyme, the technique proposed by GOTTBIEB AXD \VAHLn was applied to the polarization measurements. Accordingly, the parameter P was determined at constant temperature (25 °) and the viscosity of the medium was changed by addition of sucrose. Under this new set of experimental conditions, the plot of I / P - - 1 / 3 vs. T/r 1 shows a downward curvature at low T/r~ values. This type of curvature m a y occur whenever more than one relaxation time is present. Following the theoretical treatment proposed by MEMMINGr2 a n d GOTTBIEB AND \*rAHLll, it is possible to analyze the composite depolarization of fluorescence due to the rotation of the macromolecule (ph) and to the fast rotation of a short segment of the protein to which the dye is loosely bound (ps). If Oh > ps then Eqn. 2 can be applied to the analysis of the polarization data.
1 1 (1 -
p0-o
1'() . -
1
') (-'!
,
In this equation the parameter/3 is a function of the angles 81, 82 between the axis of rotation and the oscillators of emission and absorption, respectively. The rotational relaxation time ph and ps are related to the temperature and viscosity of the medium: 3v
K1T
3v
K2T
Oh
tj
ps
~]
(3)
Eqn. 2 adequately represents the experimental results obtained in sucrose solutions provided the parameters K 1 , K 2 and fl have the following values" K t 3.7" IO-6, K2 ~ 5" IO-5 and fl = o.8. The calculated relaxation times are included in Table II. The most salient feature of these results is that the pH of the medium has little effect on both ph and ps. Furthermore, the relaxation times pla and ps differ by more than Io-fold and this exact difference was reproducible in a number of different measurements. The shortest relaxation time m a y be tentatively assigned to a rotating unit of low molecular weight, presumably a segment of tile protein (mol. wt. 5ooo) to which the fluorophor is attached. The longest relaxation time, on the other hand, reflects the rotation of the enzyme in solution. A relaxation time of this order of magnitude (13o nsec) is in conformity with that expected for all ellipsoid of approx, mol. wt. 9oooo (axial ratio 4) la. Since a molecular weight of this order of magnitude (IOOooo) has been previously determined by equilibrium ultracentrifugation s, it is concluded that the quaternary structure of the protein is not affected at concentrations approaching those used in the enzymatic assays. ACKNOWLEDGEMENTS
The author wishes to thank Dr. T. SALO for helpful advice in the sucrose density gradient experiments. The technical assistance of M. BATTISTAis acknowledged. This research was supported by a grant from the Atomic Energy Commission (AT 4o1-3553). Biochim. Biophys. Acta, 147 (1967) 511-517
RELAXATION TIME OF ASPARTATE AMINOTRANSFERASE
517
REFERENCES i R. C. HUGHES, W. T. JENKINS AND E. H. FISHER, Proc. Natl. Acad. Sei. U.S., 48 (1962) 16I 5. 2 C. TURANO, A. GUARTOSlO, F. RlVA AND P. VECCHINI, Chem. Biol. Aspects Pyridoxat Catalysis, Proc. Syrup. 1.U.B. Rome, I962, P e r g a m o n Press, London, 1965, p- 149. 3 ~,V. T. JENKINS, D. A. YPHANTIS AND J. ~vV. SIZER, jr. Biol. Chem., 234 (1959) 51. 4 J. E. CHURCHlCH AND L. HARPRING, Biochim. Biophys. Acta, lO 5 (1965) 5755 G. WEBER AND F. W. TEALE, Trans. Faraday Soc., 53 (1967) 646. 6 R. F. STEINER AND A. J. McLISTER, J. Polymer Sci., 24 (1957) lO5. 7 R. G. MARTIN AND B. N. AMES, J. Biol. Chem., 236 (196o) 1372. 8 J. W. SIZBR AND W. T. JENKINS, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, VoI. 5, Academic Press, New York, 1962, p. 677. 9 J. E. CHURCHICH, Bioehim. Biophys. Aeta, 65 (1962) 349. IO O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . I I Y. GOTTBIEB AND P. WAHL, J. Chim. Phys., 6o (1963) 849. 12 R. MEMMING, Z. Physiol. Chem., 28 (1961) 168. 13 G. WEBER, Biochem. J., 51 (1952) 145. 14 A. J. SOPHIANOPOULOS, C. K. RHODES, C. N. HOLCOMB AND K. E. VAN HOLOE, J. Biol. Chem., 237 (1962) 11o 7 . 15 K. DALZlEI. AND A. EHRENBERG, Acla Chem. Scan&, 12 (1958) 465 . 16 E. L. KUFF, G. H. HOGEBOOM, M. J. STRIEBICH, jr. Biol. Chem., 212 (1955) 439.
Biochim. Biophys. Acta, 147 (1967) 511-517