Estimation of state and amount of phenylalanine residues in proteins by second derivative spectrophotometry

120

Biochimica et Biophysica Acta, 5 8 0 ( 1 9 7 9 ) 1 2 0 - - 1 2 8 © 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

BBA 38274

ESTIMATION OF STATE AND AMOUNT OF PHENYLALANINE RESIDUES IN PROTEINS BY SECOND DERIVATIVE SPECTROPHOTOME TRY

TETSUO ICHIKAWA a and HIROSH! TERADA b,,

a R/D Engineering Department, Analytical Instruments Division, Shimadzu Seisakusho Ltd., Nakagyo-ku, Kyoto 604 and b Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770 (Japan) (Received F e b r u a r y 2 7 t h , 1 9 7 9 )

Key words: Phenylalanine assay; Lysozyme; Ribonuclease; Insulin; Serum albumin; Second derivative spectrophotometry

Summary The second derivative absorption spectra of serum albumin, insulin, ribonuclease and lysozyme were measured under various conditions to determine the state and amount of their phenylalanine residues. The second derivative spectra of these proteins were very similar to that of phenylalanine in the region between 245 and 270 nm where tryptophan and tyrosine residues caused no appreciable interference. Denaturation of proteins with urea or guanidine hydrochloride caused decrease in the intensity of the second derivative spectra, but scarcely affected the positions of peaks and troughs. The amounts of phenylalanine residues in proteins calculated from the second derivative spectra of denatured proteins coincided well with those reported in the literature. The states of the phenylalanine residues in the proteins could be deduced from the change in optical intensity on denaturation.

Introduction

Conformational changes of proteins induced by solvent perturbation or denaturation have been studied extensively by difference absorption spectrophotometry. In the difference spectra of proteins, however, changes in the * To whom correspondence should be addressed,

121 spectral properties of phenylalanine residues are hardly detectable because they are masked by the strong overlapping absorbances of tyrosine and tryptophan, which have much larger molar extinction coefficients than that of phenylalanine. From studies on the first derivative spectra of various proteins, such as insulin, ribonuclease A, lysozyme, a-chymotrypsin and collagen, Matsushima et al. [1] and Inoue et al. [2] concluded that the first derivative spectrum of a protein could be very useful in deducing the fine spectral properties of aromatic amino acid residues, although the spectrum of phenylalanine was still affected by those of tyrosine and tryptophan. Recently we found from studies on the spectra of solutions containing various amounts of tyrosine, tryptophan and phenylalanine that the second derivative spectra of tyrosine and tryptophan have no influence at all on the spectrum of phenylalanine between 245 and 270 nm, indicating that second derivative spectrophotometry might be a very good method for both qualitative and quantitative measurements of the state of phenylalanine residues in proteins [3]. This paper reports studies on the second derivative spectra of several proteins with various aromatic amino acid contents in the native and denatured states to see whether the second derivative spectrophotometry could actually be used to determine the state and amount of phenylalanine residues in proteins. Materials and Methods N-Acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt, lot no. 73C-2010) and insulin (bovine pancreas, crystalline, lot no. 24C-3130) were purchased from Sigma Chemical Co., St. Louis (U.S.A.), lysozyme (white egg, crystalline, lot no. 267-9) and ribonuclease (bovine pancreas, crystalline, lot no. 316-7) were from P-L Biochemicals Inc., Milwaukee (U.S.A.), and serum albumin (bovine, crystalline, lot no. 3701) was from Nutritional Biochemicals Corp., Cleveland (U.S.A.). Urea, guanidine hydrochloride, 1-propanol and ethylene glycol, all of the highest purity available, were obtained from Wako Chemical Industries Ltd., Osaka (Japan) and used without further purification. The concentrations of Ac-Phe-OEt and proteins were determined spectrophotometrically using the following molar extinction coefficients: 1.97 • 102 at 257.4 nm for Ac-Phe-OEt [4], 6.1 • 10 a at 278 nm for insulin [5], 3.88 • 104 at 281 nm for lysozyme [6], 9.8 • 103 at 277.5 nm for ribonuclease [7] and 4.6 • 104 at 278 nm for serum albumin [8]. Solutions of Ac-Phe-OEt and proteins were prepared just before experiments. Stock solutions of 8.5 M urea and 7.0 M guanidine hydrochloride in 0.1 M potassium phosphate buffer (pH 7.0 or pH 8.0) were prepared just before use. Proteins were denatured by treating them in solution with urea or guanidine hydrochloride for 24 h at 8 ° C. Absorption spectra were measured in a Shimadzu recording spectrophotometer, model UV-300. Derivative absorption spectra were measured using a derivative attachment, model DES-I, connected with the spectrophotometer. The output signals from the spectrophotometer were converted electrically to the derivative analog signals. The derivative wavelength difference, AX, was 1 nm as described previously [3].

122 Results

Fig. 1 shows the absorption spectrum (curve A) and the second derivative spectrum (curve B) of the N-acetyl ethyl ester of phenylalanine (Ac-Phe-OEt) in phosphate buffer of pH 7.0. Characteristic absorption bands of phenylalanine are observed in both spectra between 245 and 270 nm. There are peaks in the second derivative spectrum of Ac-Phe-OEt at 249, 256, 260, 262, 266 and 270 nm, and troughs at 247, 252, 258, 261,264 and 268 nm, as reported previously [3]. In 8 M urea (curve C) the positions of the peaks and troughs in the second derivative spectrum of Ac-Phe-OEt are similar to those in phosphate buffer, but the absorbances are smaller. Measurement of the second derivative spectrum of Ac-Phe-OEt in various concentrations of urea showed that the absorbances of all spectral bands became smaller as the concentration of urea increased. Similar results were obtained on the spectrum of phenylalanine in guanidine hydrochloride. However, the second derivative spectrum of Ac-Phe-OEt in a solvent of low polarity, such as 90% 1-propanol or 90% ethylene glycol, showed intensified absorbances with little change in the positions of positive and negative peaks relative to those in phosphate buffer. The differences between the absorbances of various peaks and troughs, A(d2e/dk2), in the second derivative spectra of phenylalanine under various conditions are listed in Table I. The second derivative spectra of the esters of tyrosine and tryptophan in phosphate buffer of pH 7.0 showed similar values to those reported previously [3]: kmax at 279 and 288 nm and ~t~ough at 276 and 283 nm for the ester of tyrosine, and kmax at 275, 279, 286 and 294 nm and ktrough at 273, 278, 282 and 289 nm for the ester of tryptophan. The second derivative spectra of tyrosine and tryptophan showed no absorbance between 245 and 270 nm. To examine the availability of second derivative spectrophotometry for determining the state of phenylalanine residues in proteins containing tyrosine and tryptophan, we used 4 proteins in this study: insulin, ribonuclease, lysozyme and serum albumin.

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123 T A B L E I" D I F F E R E N C E S B E T W E E N P E A K S A N D T R O U G H S , A ( d 2 e / d k 2 ) , IN T H E S E C O N D D E R I V A T I V E SPECTRUM OF P H E N Y A L A N I N E U N D E R V A R I O U S C O N D I T I O N S Medium

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Fig. 2 shows the absorption spectrum and the second derivative spectrum of insulin, which has no tryptophan residues [9]. In the absorption spectrum of insulin in phosphate buffer of pH 8.0 (curve A), the absorption bands due to phenylalanine residues are almost completely masked by the strong bands of tyrosine residues in the region of 240 to 270 nm. The second derivative spectrum in phosphate buffer (curve B) shows 8 characteristic peaks and troughs between 245 and 300 nm: peaks at 250, 256,260, 263, 267, 271,281 and 289 nm, and troughs at 248, 253, 258, 261, 264, 268, 277 and 284 nm. From the second derivative spectra of phenylalanine and tyrosine, the first 6 peaks and troughs are concluded to be due to phenylalanine residues, and the last 2 A E

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peaks and troughs to tyrosine residues. Thus the strong absorbances in the second derivative spectrum of insulin in the region above 272 nm are ascribed to the large molar extinction coefficients of tyrosine. The positions of the peaks and troughs in the spectrum of insulin are almost the same (within 1 nm deviation) as those of phenylalanine and tyrpsine, indicating that the absorbances of tyrosine do not affect those of phenylalanine residues in the second derivative spectrum of insulin. The second derivative spectrum of insulin in 8 M urea (curve C) is essentially similar to that in phosphate buffer, but the positions of some of the peaks and troughs are slightly shifted to shorter wavelengths, and the intensities are less than those of native insulin. As shown in Fig. 3, similar spectral properties to those of insulin are observed under various conditions for the spectrum of ribonuclease, which also does not contain any tryptophan residues [10]. Fig. 4 shows the spectra of lysozyme, which contains 3 tyrosines, 6 tryptophans and 3 phenylalanines [11]. The absorption spectrum of lysozyme in i

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125

phosphate buffer of pH 7.0 (curve A) exhibits strong bands of tryptophan and tyrosine residues in the region between 270 and 300 nm, with scarcely detectable spectral bands of phenylalanine residues. There are 9 maxima at 249, 254, 259, 262, 266, 270, 277, 287 and 294 nm and minima at 247, 252, 258, 261, 264, 268, 273, 283 and 290 nm in the second derivative spectrum of lysozyme in phosphate buffer of pH 7.0 (curve B). Strong absorbances due to tryptophan and tyrosine in the region above 271 nm are noted. Treatment of native lysozyme with 8 M urea causes a slight red-shift, decrease in intensities of absorbances below 270 nm and increase in those above 270 nm (curve C). The spectrum of lysozyme denatured with 6 M guanidine hydrochloride (curve D) shows a marked decrease in the absorption intensities with little change in the positions of peaks and troughs, as compared with the spectrum of native lysozyme in all wavelength regions. The difference in the second derivative spectrum of denatured lysozyme from that of native lysozyme is greater with guanidine hydrochloride as denaturating reagent than with urea, indicating that the latter does not cause complete denaturation. Bovine serum albumin contains all types o f aromatic amino acid residues, but unlike the spectra of most other proteins its absorption spectrum at pH 7.0 shows weak bands of phenylalanine residues between 240 and 270 nm which are not completely hidden by th~ absorptions of tyrosine and tryptophan (Fig. 5, curve A), since the contents of phenylalanine residues in serum albumin are higher (26 residues) than those of tyrosine (19 residues) and tryptophan (2 residues) [12]. The second derivative Spectrum of native serum albumin clearly shows characteristic bands due to phenylalanine residues (curve B), with kmax at 2 5 1 , 2 5 7 , 261, 263, 267 and 271 nm and k=ough at 248, 253, 259, 262, 265 and 269 nm. Above 272 nm, the spectral barlds of tyrosine and tryptophan residues are also observed with kma x at 2 7 5 , 2 8 3 , 2 9 0 and 294 nm, and ~tzough at 274, 280, 286 and 291 nm. Denaturation of serum albumin with 8 M urea resulted in greater changes in the second derivative spectrum (curve C) than those observed for insulin, ribonuclease and lysozyme: the kmax and htruugh lh

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126 shift to shorter wavelengths, especially in the region where spectral bands due to tryptophan and tyrosine are dominant, and the intensities of the bands in all regions decrease markedly. Discussion

Below 270 nm, the second derivative spectra of insulin, ribonuclease, lysozyme and serum albumin were essentially the same as the second derivative spectrum of phenylalanine, irrespective of the contents of aromatic amino acid residues in these proteins, indicating that second derivative spectrophotometry is very useful for study of the phenylalanine residues in proteins. In the second derivative spectrum of proteins, characteristic spectral bands due to phenylalanine are generally observed between 245 and 270 nm: kmax at 249, 256, 260, 262, 266 and 270 nm and ~trough at 247, 252, 258, 261,264 and 268 nm, within 1 nm deviation. Denaturation of proteins with urea or guanidine hydrochloride induced little shift in the ~max and ~trough due to phenylalanine (mostly only 1 nm shift to shorter wavelength), but caused marked decrease in the absorbances. This indicates that change in conditions around phenylalanine residues in proteins is mainly reflected, not in the positions of peaks and troughs, but in the intensities of the bands. Sometimes change in the environment around chromophoric residues in proteins caused by denaturation or solvent perturbation has been discussed in terms of shift in the peaks of the absorption spectrum of proteins [13]. However, in this study there was an instrumental limitation to determining the positions of the peaks and troughs exactly enough to distinguish a difference of 1 nm in the second derivative spectrum. Thus attention was focused on change in the intensities of bands in the second derivative spectra of proteins. Table I lists the differences in the molar extinction coefficients, A(d2e/d~2), between various peaks * and troughs in the second derivative absorption spectra of Ac-Phe-OEt under various conditions. The values of A(d2e/dk 2) consistently decrease as the concentration of urea increases and the values in the presence of 6 M guanidine hydrochloride are smaller than those in aqueous solution. On the other hand, the A(d2e/dk :) values for each pair of positive and negative peaks in 1-propanol and ethylene glycol are greater than those in aqueous solution. These results indicate that all the electrical fine structures (vibrational structures) of phenylalanine are influenced by change in the environment in a similar way. Thus from the difference in absorbance between a certain pair of positive and negative peaks in the second derivative spectrum it is possible to obtain information on the environment and location of phenylalanine residues in a protein, if the pair chosen is sensitive enough to the environment for use in the evaluation. As shown in the table, values for the difference in optical intensity of the wavelength pair D--C (262--264 nm) are greatest, suggesting that this wavelength pair is the most sensitive. However, * Since t h e p o s i t i o n s o f p o s i t i v e a n d negative p e a k s in s e c o n d d e r i v a t i v e s p e c t r a of p h e n y l a l a n i n e a n d p r o reins w e r e slightly d e p e n d e n t o n t h e p r o p e r t i e s o f t h e m e d i u m , t h e i r m a i n p e a k s are i n d i c a t e d as p e a k s A - - E in T a b l e I. W h e n t h e p o s i t i o n s o f t h e s e p e a k s are expressed in terms o f w a v e l e n g t h in the t e x t a n d in the tables, t h e y are t h o s e o b s e r v e d w i t h p h e n y l a l a n i n e in 0.1 M p h o s p h a t e buffer, p H 7.0, unless otherwise noted.

127 TABLE

II

AVERAGE ABSORBANCE DIFFERENCE OF SECOND DERIVATIVE SPECTRA OF PROTEINS AND AVERAGE NUMBER OF PHENYLALANINE RESIDUES IN PROTEINS CALCULATED FROM SECOND DERIVATIVE SPECTRA a

Average a b s o r b a n c e difference,

A(d2A/d~2)/Cp( 1 0 1 5

Serum albumin

Insulin Lysozyme Ribonuclease

Number

of

In native

In denatured

phenylalanine residues per protein

protein

protein

molecule b

88.16 ± 0.15 9.73 + 0.12 22.19 ± 0.30

70.46 8.13 7.83 20.87 8.13

9.42 ± 0.12

M-1 . cm-2)

± ± ± ± ±

0.13 0.08 0.23 0.29 0.11

c c d e c

Values in literature Phe

Tyr

Trp

26.0 + 0.05 3.0 ± 0.03 3.0 ± 0.09

26 3 3

19 4 3

2 [12] 0 [9] 6 [11]

3.0 ± 0.04

3

6

0 [10]

a Determined f r o m the a b s o r b a n c e difference o f the w a v e l e n g t h pair 2 6 6 - - 2 6 8 n m ( B - - A ) . See Table I. V a l u e s in the table are m e a n s for at least 5 runs. b D e t e r m i n e d according t o E q n . 1 using values for p r o t e i n s denatured b y 8 M urea, e x c e p t w i t h l y s o z y m e , w h e r e the value after d e n a t u r a t i o n w i t h 6 M guanidine h y d r o c h l o r i d e is used. c In 8Murea. d I n 6 M guanidine h y d r o c h l o r i d e .

it is desirable not to choose a wavelength pair shorter than about 260 nm, since the effect of satellite components * due to tyrosine and tryptophan on second derivative spectrum of phenylalanine is very great in this region, especially for proteins, such as lysozyme, containing little phenylalanine relative to tyrosine and tryptophan. Thus for estimation of the state of phenylalanine residues in proteins, it is better to choose the wavelength pair B--A (266--268 nm), although the difference in absorbance of this wavelength pair is less. Table II summarizes the values of average absorbance difference per protein molecule, ,h(d2A/dk2)/Cp, between positive and negative peaks of B--A (266--268 nm) due to proteins in the native and denatured states (Cp: concentration of protein). In all cases values with native protein are greater than those with denatured. Since, as shown in Table I, the optical intensities of phenylalanine become great in hydrophobic media, the results in Table II indicate that the phenylalanine residues in these proteins are in a hydrophobic environment, that is buried in the interior of protein molecules as observed with X-ray diffraction and the solvent perturbation studies [14--18]. It is noted in Table II that the absorb0nce difference of lysozyme becomes only slightly smaller on denaturation with 8 M urea, while it decreases greatly on treatment with 6 M guanidine hydrochloride. This is compatible with the finding that lysozyme is not completely denatured by 8 M urea, but fully denatured by 6 M guanidine hydrochloride [19,20]. o f a certain c o m p o n e n t A c o n t a i n s an a d d i t i o n a l c o m p o n e n t B, the optical intensity o f c o m p o n e n t A in the s e c o n d derivative s p e c t r u m will be greatly i n f l u e n c e d b y that o f c o m p o n e n t B in the w a v e l e n g t h region w h e r e the curvature o f the n o r m a l a b s o r p t i o n s p e c t r u m o f c o m p o n e n t B is great. T h u s in this region it is very difficult t o d e t e r m i n e a c c u r a t e l y the optical i n t e n s i t y o f c o m p o n e n t A in the s e c o n d derivative s p e c t r u m . We d e n o t e t h e e f f e c t o f the a b s o r p t i o n s p e c t r u m o f o t h e r c o m p o n e n t , s u c h as c o m p o n e n t B, in this w a v e l e n g t h region 'the e f f e c t o f s a t e l l i t e c o m p o n e n t s ' . Theoretical cons i d e r a t i o n o f this effect will be r e p o r t e d in a s u b s e q u e n t paper.

* When a solution

128

Using the values of the absorbance difference, A(d2A/dk2)/Cv, of the fully denatured and unfolded protein listed in Table II, the average number of phenylalanine residues in proteins (n) can be calculated according to Eqn. 1, where l is the optical path length (=1 cm). 1 n-Cp.l

A(d2A/dk 2) A(d:e/dk 2)

(1)

For the calculation, the value of A(d2e/dk 2) with phenylalanine in 8 M urea (with insulin, serum albumin and ribonuclease) or in 6 M guanidine hydrochloride (with lysozyme) at the wavelength pair B--A (266--268 nm) listed in Table I is used. The numbers of phenylalanine residues in proteins, calculated by Eqn. 1 and shown in Table II, coincide quite well with the values determined by amino acid analysis [9--12]. Thus from the second derivative absorbances of proteins in 8 M urea or 6 M guanidine hydrochloride, the average number of phenylalanine residues can easily be determined without complicating effects of other aromatic amino acid residues, as proposed by Balestrieri et al. [21], if an appropriate wavelength pair is chosen. Balestrieri et al. [21] calculated the amounts of phenylalanine residues in various proteins from the difference of the second derivative absorbances at 257 and 259 nm, where the effect of satellite components could be rather great. The discrepancies between their values and the values in the literature could be due to the effect of satellite components and also to the fact that resolution in the second derivative spectra is not high. Our results clearly show that second derivative spectrophotometry is very useful for obtaining information about the state and amount of phenylalanine residues in proteins. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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