Biochimica et Biophysica Acta,494 ( 1 9 7 7 ) 2 6 7 - - 2 7 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
B B A Report BBA 31228
SECOND DERIVATIVE SPECTROPHOTOMETRY AS AN EFFECTIVE TOOL FOR EXAMINING PHENYLALANINE RESIDUES IN PROTEINS
TETSUO
ICHIKAWA
a and H I R O S H I T E R A D A
b,*
aScientificInstrument Plant, Shimadzu Seisakusho, Co., Nakagyo-ku, Kyolo 604 and b Faculty of Pharmaceutical Sciences, Universityof Tokushima, Shomachi-1, Tokushima 770 (Japan) (Received June 6th, 1977)
Summary The second derivative absorption spectra of N-acetyl ethyl esters of phenylalanine, tyrosine and tryptophan, as models of the aromatic amino acid residues in proteins, were measured. The second derivative spectra of tyrosine and tryptophan were found to have no influence on the spectrum of phenylalanine over the range of 245 to 270 nm, where characteristic absorbance bands of phenylalanine were observed. Thus the second derivative spectrum is a good tool for examining the optical properties of phenylalanine residues in proteins.
Tyrosine, tryptophan and phenylalanine residues in proteins have characteristic absorption bands in the ultraviolet region. Since the changes in the absorption spectra of proteins induced by solvent perturbation or denaturation are in many cases very small, difference spectra of proteins have been used extensively to characterize the states of these three amino acid residues in proteins. However, changes in the spectral properties of phenylalanine can hardly be detected because they are masked by the strong overlapping absorbances of tyrosine and tryptophan. Thus most spectrophotometric studies on the states of amino acid residues in protein molecules have been focused on tyrosine and tryptophan residues [1--3]. French and Church [4] suggested the possibility of using derivative absorption spectrophotometry for the detection of weak bands masked by strong ones. Recently, Matsushima et al. [5] and Inoue et al. [6] reported the application of first derivative spectrophotometry (the derivative of absorbance, A, or the molar extinction coefficient, e, with respect to wavelength, k: dA/d), or de/dk) to the analysis of phenylalanine residues in various proteins. However, the effects of the absorbances of tyrosine and tryptophan residues on the ab*To whom co~espondence should be addressed.
268 sorbance of phenylalanine residues cannot be eliminated completely by this method. This paper deals with the availability of second derivative spectrop h o t o m e t r y {d2A/d)` 2 or d2e/d)` 2 ) for examining the optical properties of phenylalanine and completely excluding the effects of tyrosine and tryptophan. In this w o r k we used N-acetyl ethyl esters of aromatic amino acids as models of amino acid residues in proteins. The N-acetyl ethyl esters of L-phenylalanine (Ac-Phe-OEt), L-tyrosine (Ac-Tyr-OEt) and L-tryptophan (Ac-Trp-OEt} were purchased from Sigma Chemical Co., Saint Louis (U.S.A.), E. Merck AG, Darmstadt {G.F.R.) and T o k y o Kasei Kogyo Co., T o k y o (Japan), respectively. These amino acid derivatives were dissolved in 0.1 M potassium phosphate buffer (pH 7.0) just before the experiments and their concentrations were determined spectrophotometrically using the following molar extinction coefficients: 197 for Ac-PheOEt at 257.4 nm [7], 1420 for Ac-Tyr-OEt at 274.5 nm [3] and 5550 for Ac-Trp-OEt at 279.9 nm [3]. Absorption spectra were measured in a Shimadzu recording spectrophotometer, model UV-300. The derivative absorption spectra were measured using a derivative attachment, model DES-l, connected with the spectrophotometer. The o u t p u t signals from the spectrophotometer were converted electrically into the derivative analog signals, and these gave a flat base line. The derivative wavelength difference 4)` = I nm was selected to obtain good resolution and a low noise level. As has been reported by Herskovits and Sorensen [ 1], the absorption spectrum of an aqueous solution {pH 7.0) of Ac-Trp-OEt with a )'max of 279.9 nm was similar to that of Ac-Tyr-OEt with a Xmax of 274.5 nm. However, the absorption spectrum of Ac-Phe-OEt showed several sharp peaks in wavelength region of 240 to 270 nm, and differed distinctly from those of Ac-Trp-OEt and Ac-Tyr-OEt. When the spectrum of a solution containing 7.70- 10 -4 M Ac-Phe-OEt, 3 . 0 1 . 1 0 -4 M Ac-Tyr-OEt and 1.46. ~0 -4 M Ac-Trp-OEt at pH 7.0 was measured, the characteristic absorbance bands of phenylalanine between 240 and 270 nm were completely masked b y the strong bands of tryptophan and tyrosine, although their concentrations were less than half that of phenylalanine, and the overall spectrum was very similar to those of proteins, such as serum albumin [8] and ribonuclease [9]. Fig. 1 shows the first derivative absorption spectra (dA/d)`) of the aromatic amino acids. The derivative spectrum of Ac-Trp-OEt (Curve A) shows three troughs in the region of 270 to 300 nm and a broad band between 240 and 270 nm, and that of Ac-Tyr-OEt (Curve B) has t w o troughs and a broad band in the respective wavelength regions. The derivative spectrum of Ac-Phe-OEt (Curve C) has no first derivative c o m p o n e n t (dA/dX = 0) between 270 and 300 nm, and six negative peaks in the shorter wavelength region, as reported by Matsushima et al. [ 5]. It is clear from Fig. 1 that the optical effects of tyrosine and tryptophan on the spectrum of phenylalanine are reduced, but not completely eliminated, in the first derivative spectrum. Fig. 2 shows the secondary derivative absorption spectra (d2A/d)` ~) of three aromatic amino acids. The second derivative spectra of tyrosine and tryptophan are completely flat between 245 and 270 nm, where the spectrum
269 ,
,
,
I /' ,~ fl t+,~%Li~_
240
i
jj,¸,J
C \/ . . . . .
260
280
300
320
Wovelengf h (nm) Fig. 1. T h e fi~st d e r i v a t i v e a b s o r p t i o n s p e c t r a of N - a c e t y l e t h y l e s t e ~ of t r y p t o p h ~ ( C u ~ e A), ty~osine ( C u ~ e B) ~ d p h e n y l ~ e ( C u ~ e C) d i ~ o l v e d ~ 0 . 1 M p h o s p h a t e b u f f e r of p H 7.0. C o n c e n t r a t i o ~ o f ~ i n o acids: t r y p t o ~ h ~ , 1.51 "10 -4 M, t y r o s ~ e , 3.46 • 10 ~ M; p h e n y l ~ i n e , 3 . 5 8 • 10- 3 M.
~
~
~/
~1
~
~
'!~
/'
,,
oo
~ ~, ~ ~,~ t~ ~ ~'
~,,~: l 2 20
v
~
~':~-----
~ 'h' , I ~ ' ,
~,~~, , 240
~ 260
, 280
, 500
Wovelength ( n m ) Fig, 2. T h e s e c o n d d e ~ v a t i v e a b s o ~ t i o n s p e c t r a o f the N - a c e t y l e t h y l esters of 1.51 • 10 ~ M t ~ P t o p h ~ ( C u ~ e A), 3.46 • 10 -4 M t y r o s ~ e ( C u ~ e B) ~ d 3 . 5 8 • 10 z M p h e n y l ~ i n e ( C u ~ e C) in 0.1 M p h o ~ h a t e b u f f e r of p H 7.0,
of phenylalanine shows its characteristic spectral bands (~max : 249, 256, 260, 262, 266 and 270 nm, and ~.trough: 2 4 7 , 2 5 2 , 2 5 8 , 2 6 1 , 2 6 4 and 268 nm between 245 and 270 nm). Thus we can analyse the state of phenylalanine even in the presence of t r y p t o p h a n and tyrosine by using the second derivative absorption spectrum. This was confirmed by the results in Table I, where the A(d2e/d~ 2 ) values (the difference in d2e/d~ ~ values between the strong positive peak at 262 nm and the most intensive negative peak at 264 nm) of 7.70- 10 -4 M solution of Ac-Phe-OEt in the presence of various concentrations of Ac-Trp-OEt and Ac-Tyr-OEt at pH 7.0 are listed. They consistently show the same value. In the table, the molar ratios of amino acids were taken so as to be similar to those in various proteins, such as insulin (experiment d), ribonuclease (e), ovalbumin (f), serum albumin (g) and ~-lactoglobulin (g). As shown in Fig. 3, the value of A (d2A/dk: ) of phenylalanine changes linearly with the concentration of phenylalanine and the slope of the straight line (~ A (d~e/d), ~)) is 4.4- 10 is M-1. cm-3, which is the same as the value of A (d 2e/dk :) for Ac-Phe-OEt listed in Table I.
270
006
,
,
,
c .~~ 0 0 4 ,~
OO2
0 0
I
~
4
8
12
Concentrution of Ac-Phe-OEt xlO"(M)
Fig. 3. L i n e a r r e l a t i o n s h i p b e t w e e n A ( d ~ A / d k ~) a n d t h e c o n c e n t r a t i o n o f N - a c e t y l e t h y l e s t e r o f p h e n y l a l a n i n e i n 0.1 M p h o s p h a t e b u f f e r o f p H 7.0. A ( d ~ A / d k 2 ) : d i f f e r e n c e b e t w e e n d 2 A / d k ~ v a l u e s a t 2 6 2 r a n and at 264 nm. TABLE I THE SECOND DERIVATIVE (A(d~e/d~)) OF THE N-ACETYL ETHYL ESTER OF PHENYLA L A N I N E IN T H E P R E S E N C E O F T H E E S T E R S O F T Y R O S I N E A N D T R Y P T O P H A N A T p H 7 . 0 Expt.
a b c d e f g
C o n c e n t r a t i o n ( 1 0 -4 M) o f N - a c e t y l e t h y l e s t e r o f
A(d2e/dk~) *
Phenylalanine
Tyrosine
Tryptophan
(102 s M - ~ • c m 3)
7.70 7.70 7.70 7.70 7.70 7.70 7.70
0 3.01 6.03 9.04 12.06 3.01 9.04
0 0 0 0 0 1.46 2.18
4.4 4.4 4.4 4.2 4.4 4.3 4.3
* D i f f e r e n c e b e t w e e n d~e/dA 2 v a l u e s a t 2 6 2 n m a n d 2 6 4 n m .
The results in Figs. 2 and 3 and Table I indicate that the second derivative spectrophotometry is very good method for both qualitative and quantitative measurements of the state of phenylalanine residues in protein molecules. We are grateful to Mr. Y. Tsunazawa for his valuable suggestions. References 1 2 3 4 5 6 7
H e r s k o v i t s , T.T. a n d S o r e n s e n , S.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 2 5 2 3 - - 2 5 3 2 H e r s k o v i t s , T.T. a n d S o r e n s e n , S.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 2 5 3 3 - - 2 5 4 2 Solli, J . N . a n d H e r s k o v i t s , T.T. ( 1 9 7 3 ) A n a l . B i o c h e m . 5 4 , 3 7 0 - - 3 7 8 F r e n c h , C.S. a n d C h u r c h , A . B . ( 1 9 5 5 ) C a r n e g i e Inst. W a s h . Y e a r B o o k 54, 1 6 2 - - 1 6 5 M a t s u s h i m a , A., I n o u e , Y. a n d Shibats~ K. ( 1 9 7 5 ) A n a l . B i o c h e m . 6 5 , 3 6 2 - - 3 6 8 I n o u e , Y , M a t s u s l l i m a , A. a n d S h i b a t a , K. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 7 9 , 6 5 3 - - 6 5 7 G r a t z e r , W.B. ( 1 9 7 0 ) in H a n d b o o k o f B i o c h e m i s t r y : S e l e c t e d D a t a f o r B i o c h e m i s t r y ( S o b e r , H . A . , ed.), pp. B 74--77, Chemical Rubber Co., Cleveland 8 T a n f o r d , C. ( 1 9 5 0 ) J. A m . C h e m . S o c . 72, 4 4 1 ~ 4 5 1 9 T a n f o r d , C., H a u e n s t e i n , .I.D. a n d R a n d s , D . G . ( 1 9 5 6 ) .I. A m . C h e m . S o c . 6 4 0 9 - 6 4 1 3
B B A Report BBA 31228
SECOND DERIVATIVE SPECTROPHOTOMETRY AS AN EFFECTIVE TOOL FOR EXAMINING PHENYLALANINE RESIDUES IN PROTEINS
TETSUO
ICHIKAWA
a and H I R O S H I T E R A D A
b,*
aScientificInstrument Plant, Shimadzu Seisakusho, Co., Nakagyo-ku, Kyolo 604 and b Faculty of Pharmaceutical Sciences, Universityof Tokushima, Shomachi-1, Tokushima 770 (Japan) (Received June 6th, 1977)
Summary The second derivative absorption spectra of N-acetyl ethyl esters of phenylalanine, tyrosine and tryptophan, as models of the aromatic amino acid residues in proteins, were measured. The second derivative spectra of tyrosine and tryptophan were found to have no influence on the spectrum of phenylalanine over the range of 245 to 270 nm, where characteristic absorbance bands of phenylalanine were observed. Thus the second derivative spectrum is a good tool for examining the optical properties of phenylalanine residues in proteins.
Tyrosine, tryptophan and phenylalanine residues in proteins have characteristic absorption bands in the ultraviolet region. Since the changes in the absorption spectra of proteins induced by solvent perturbation or denaturation are in many cases very small, difference spectra of proteins have been used extensively to characterize the states of these three amino acid residues in proteins. However, changes in the spectral properties of phenylalanine can hardly be detected because they are masked by the strong overlapping absorbances of tyrosine and tryptophan. Thus most spectrophotometric studies on the states of amino acid residues in protein molecules have been focused on tyrosine and tryptophan residues [1--3]. French and Church [4] suggested the possibility of using derivative absorption spectrophotometry for the detection of weak bands masked by strong ones. Recently, Matsushima et al. [5] and Inoue et al. [6] reported the application of first derivative spectrophotometry (the derivative of absorbance, A, or the molar extinction coefficient, e, with respect to wavelength, k: dA/d), or de/dk) to the analysis of phenylalanine residues in various proteins. However, the effects of the absorbances of tyrosine and tryptophan residues on the ab*To whom co~espondence should be addressed.
268 sorbance of phenylalanine residues cannot be eliminated completely by this method. This paper deals with the availability of second derivative spectrop h o t o m e t r y {d2A/d)` 2 or d2e/d)` 2 ) for examining the optical properties of phenylalanine and completely excluding the effects of tyrosine and tryptophan. In this w o r k we used N-acetyl ethyl esters of aromatic amino acids as models of amino acid residues in proteins. The N-acetyl ethyl esters of L-phenylalanine (Ac-Phe-OEt), L-tyrosine (Ac-Tyr-OEt) and L-tryptophan (Ac-Trp-OEt} were purchased from Sigma Chemical Co., Saint Louis (U.S.A.), E. Merck AG, Darmstadt {G.F.R.) and T o k y o Kasei Kogyo Co., T o k y o (Japan), respectively. These amino acid derivatives were dissolved in 0.1 M potassium phosphate buffer (pH 7.0) just before the experiments and their concentrations were determined spectrophotometrically using the following molar extinction coefficients: 197 for Ac-PheOEt at 257.4 nm [7], 1420 for Ac-Tyr-OEt at 274.5 nm [3] and 5550 for Ac-Trp-OEt at 279.9 nm [3]. Absorption spectra were measured in a Shimadzu recording spectrophotometer, model UV-300. The derivative absorption spectra were measured using a derivative attachment, model DES-l, connected with the spectrophotometer. The o u t p u t signals from the spectrophotometer were converted electrically into the derivative analog signals, and these gave a flat base line. The derivative wavelength difference 4)` = I nm was selected to obtain good resolution and a low noise level. As has been reported by Herskovits and Sorensen [ 1], the absorption spectrum of an aqueous solution {pH 7.0) of Ac-Trp-OEt with a )'max of 279.9 nm was similar to that of Ac-Tyr-OEt with a Xmax of 274.5 nm. However, the absorption spectrum of Ac-Phe-OEt showed several sharp peaks in wavelength region of 240 to 270 nm, and differed distinctly from those of Ac-Trp-OEt and Ac-Tyr-OEt. When the spectrum of a solution containing 7.70- 10 -4 M Ac-Phe-OEt, 3 . 0 1 . 1 0 -4 M Ac-Tyr-OEt and 1.46. ~0 -4 M Ac-Trp-OEt at pH 7.0 was measured, the characteristic absorbance bands of phenylalanine between 240 and 270 nm were completely masked b y the strong bands of tryptophan and tyrosine, although their concentrations were less than half that of phenylalanine, and the overall spectrum was very similar to those of proteins, such as serum albumin [8] and ribonuclease [9]. Fig. 1 shows the first derivative absorption spectra (dA/d)`) of the aromatic amino acids. The derivative spectrum of Ac-Trp-OEt (Curve A) shows three troughs in the region of 270 to 300 nm and a broad band between 240 and 270 nm, and that of Ac-Tyr-OEt (Curve B) has t w o troughs and a broad band in the respective wavelength regions. The derivative spectrum of Ac-Phe-OEt (Curve C) has no first derivative c o m p o n e n t (dA/dX = 0) between 270 and 300 nm, and six negative peaks in the shorter wavelength region, as reported by Matsushima et al. [ 5]. It is clear from Fig. 1 that the optical effects of tyrosine and tryptophan on the spectrum of phenylalanine are reduced, but not completely eliminated, in the first derivative spectrum. Fig. 2 shows the secondary derivative absorption spectra (d2A/d)` ~) of three aromatic amino acids. The second derivative spectra of tyrosine and tryptophan are completely flat between 245 and 270 nm, where the spectrum
269 ,
,
,
I /' ,~ fl t+,~%Li~_
240
i
jj,¸,J
C \/ . . . . .
260
280
300
320
Wovelengf h (nm) Fig. 1. T h e fi~st d e r i v a t i v e a b s o r p t i o n s p e c t r a of N - a c e t y l e t h y l e s t e ~ of t r y p t o p h ~ ( C u ~ e A), ty~osine ( C u ~ e B) ~ d p h e n y l ~ e ( C u ~ e C) d i ~ o l v e d ~ 0 . 1 M p h o s p h a t e b u f f e r of p H 7.0. C o n c e n t r a t i o ~ o f ~ i n o acids: t r y p t o ~ h ~ , 1.51 "10 -4 M, t y r o s ~ e , 3.46 • 10 ~ M; p h e n y l ~ i n e , 3 . 5 8 • 10- 3 M.
~
~
~/
~1
~
~
'!~
/'
,,
oo
~ ~, ~ ~,~ t~ ~ ~'
~,,~: l 2 20
v
~
~':~-----
~ 'h' , I ~ ' ,
~,~~, , 240
~ 260
, 280
, 500
Wovelength ( n m ) Fig, 2. T h e s e c o n d d e ~ v a t i v e a b s o ~ t i o n s p e c t r a o f the N - a c e t y l e t h y l esters of 1.51 • 10 ~ M t ~ P t o p h ~ ( C u ~ e A), 3.46 • 10 -4 M t y r o s ~ e ( C u ~ e B) ~ d 3 . 5 8 • 10 z M p h e n y l ~ i n e ( C u ~ e C) in 0.1 M p h o ~ h a t e b u f f e r of p H 7.0,
of phenylalanine shows its characteristic spectral bands (~max : 249, 256, 260, 262, 266 and 270 nm, and ~.trough: 2 4 7 , 2 5 2 , 2 5 8 , 2 6 1 , 2 6 4 and 268 nm between 245 and 270 nm). Thus we can analyse the state of phenylalanine even in the presence of t r y p t o p h a n and tyrosine by using the second derivative absorption spectrum. This was confirmed by the results in Table I, where the A(d2e/d~ 2 ) values (the difference in d2e/d~ ~ values between the strong positive peak at 262 nm and the most intensive negative peak at 264 nm) of 7.70- 10 -4 M solution of Ac-Phe-OEt in the presence of various concentrations of Ac-Trp-OEt and Ac-Tyr-OEt at pH 7.0 are listed. They consistently show the same value. In the table, the molar ratios of amino acids were taken so as to be similar to those in various proteins, such as insulin (experiment d), ribonuclease (e), ovalbumin (f), serum albumin (g) and ~-lactoglobulin (g). As shown in Fig. 3, the value of A (d2A/dk: ) of phenylalanine changes linearly with the concentration of phenylalanine and the slope of the straight line (~ A (d~e/d), ~)) is 4.4- 10 is M-1. cm-3, which is the same as the value of A (d 2e/dk :) for Ac-Phe-OEt listed in Table I.
270
006
,
,
,
c .~~ 0 0 4 ,~
OO2
0 0
I
~
4
8
12
Concentrution of Ac-Phe-OEt xlO"(M)
Fig. 3. L i n e a r r e l a t i o n s h i p b e t w e e n A ( d ~ A / d k ~) a n d t h e c o n c e n t r a t i o n o f N - a c e t y l e t h y l e s t e r o f p h e n y l a l a n i n e i n 0.1 M p h o s p h a t e b u f f e r o f p H 7.0. A ( d ~ A / d k 2 ) : d i f f e r e n c e b e t w e e n d 2 A / d k ~ v a l u e s a t 2 6 2 r a n and at 264 nm. TABLE I THE SECOND DERIVATIVE (A(d~e/d~)) OF THE N-ACETYL ETHYL ESTER OF PHENYLA L A N I N E IN T H E P R E S E N C E O F T H E E S T E R S O F T Y R O S I N E A N D T R Y P T O P H A N A T p H 7 . 0 Expt.
a b c d e f g
C o n c e n t r a t i o n ( 1 0 -4 M) o f N - a c e t y l e t h y l e s t e r o f
A(d2e/dk~) *
Phenylalanine
Tyrosine
Tryptophan
(102 s M - ~ • c m 3)
7.70 7.70 7.70 7.70 7.70 7.70 7.70
0 3.01 6.03 9.04 12.06 3.01 9.04
0 0 0 0 0 1.46 2.18
4.4 4.4 4.4 4.2 4.4 4.3 4.3
* D i f f e r e n c e b e t w e e n d~e/dA 2 v a l u e s a t 2 6 2 n m a n d 2 6 4 n m .
The results in Figs. 2 and 3 and Table I indicate that the second derivative spectrophotometry is very good method for both qualitative and quantitative measurements of the state of phenylalanine residues in protein molecules. We are grateful to Mr. Y. Tsunazawa for his valuable suggestions. References 1 2 3 4 5 6 7
H e r s k o v i t s , T.T. a n d S o r e n s e n , S.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 2 5 2 3 - - 2 5 3 2 H e r s k o v i t s , T.T. a n d S o r e n s e n , S.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 2 5 3 3 - - 2 5 4 2 Solli, J . N . a n d H e r s k o v i t s , T.T. ( 1 9 7 3 ) A n a l . B i o c h e m . 5 4 , 3 7 0 - - 3 7 8 F r e n c h , C.S. a n d C h u r c h , A . B . ( 1 9 5 5 ) C a r n e g i e Inst. W a s h . Y e a r B o o k 54, 1 6 2 - - 1 6 5 M a t s u s h i m a , A., I n o u e , Y. a n d Shibats~ K. ( 1 9 7 5 ) A n a l . B i o c h e m . 6 5 , 3 6 2 - - 3 6 8 I n o u e , Y , M a t s u s l l i m a , A. a n d S h i b a t a , K. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 7 9 , 6 5 3 - - 6 5 7 G r a t z e r , W.B. ( 1 9 7 0 ) in H a n d b o o k o f B i o c h e m i s t r y : S e l e c t e d D a t a f o r B i o c h e m i s t r y ( S o b e r , H . A . , ed.), pp. B 74--77, Chemical Rubber Co., Cleveland 8 T a n f o r d , C. ( 1 9 5 0 ) J. A m . C h e m . S o c . 72, 4 4 1 ~ 4 5 1 9 T a n f o r d , C., H a u e n s t e i n , .I.D. a n d R a n d s , D . G . ( 1 9 5 6 ) .I. A m . C h e m . S o c . 6 4 0 9 - 6 4 1 3
