653 Biochimica et Biophysica Acta, 379 (1975) 653--657

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

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BBA 31186 D I F F E R E N C E - D E R I V A T I V E ABSORBANCE S P E C T R O P H O T O M E T R Y AS A T E C H N I Q U E TO M E A S U R E STATE CHANGES OF P H E N Y L A L A N I N E RESIDUES IN PROTEINS

YORINAO

I N O U E a, A Y A K O

MATSUSHIMA

b

and K A Z U O S H I B A T A a

a The Institute of Physical and Chemical Research, Wako-shi, Saitama, and b Tokyo Institute of Technology, Ookayama, Tokyo, Meguroku (Japan)

(Received December 3rd, 1974)

Summary The first derivatives of difference absorbance spectra of several proteins were measured to examine the applicability of this technique as a tool to investigate state changes of phenylalanine residues in proteins. It was found by this technique that phenylalanine residues in insulin and those in lysozyme are exposed to more aqueous environment b y denaturation with guanidine hydrochloride. Heat denaturation of collagen caused similar changes of some of its phenylalanine residues. It was thus demonstrated that difference-derivative absorbance s p e c t r o p h o t o m e t r y gives the information a b o u t state changes of phenylalanine residues in native proteins, which are hardly detected b y common difference spectrophotometry.

Conformational changes of proteins on the formation of enzymesubstrate complexes or on denaturation have been a subject under extensive investigations by means of difference absorbance spectrophotometry. In the difference spectra, however, changes of weak absorbance bands are masked b y changes of strong bands in the same wavelength region. For example, small spectral changes of phenylalanine residues in a protein molecule are masked by large changes o f tyrosine and t r y p t o p h a n bands. In the present study, a simple technique previously developed to measure derivative absorbance spectra [ 1 ] was applied to measure the derivative with respect to wavelength of difference-absorbance spectra (denoted as differencederivative spectrum), with the hope that we might observe state changes of

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phenylalanine residues in proteins upon denaturation by heat or by guanidine hydrochloride. This is based on the principle that a small spectral change masked in large changes in a difference spectrum can be detected in the difference-derivative spectrum, if the derivative of this small change with respect to wavelength is appreciable. Crystalline bovine zinc insulin and lysozyme (egg white 5 times crystallized) were purchased from Shimizu Pharmaceutical Co. and Seikagaku Kogyo Co., respectively. Collagen was prepared from carp swim bladders by the m e t h o d of Gallop [2], and gelatin was obtained by heating collagen for 30 min at 50°C in acetate buffer (pH 4.8, ionic strength 0.06) by the m e t h o d of Piez, Eigner and Lewis [3]. The concentrations of amino acids and proteins were determined spectrophotometrically, assuming following values of e (molar extinction coefficient); 1.97" 102 cm -] "M-' at 257.4 nm for phenylalanine [4], 6.1 "103 cm - ' .M -~ at 278 nm for insulin [5], 3.88.104 cm -1 "M-1 at 281 nm for lysozyme [6]. The concentrations of collagen and gelatin were determined gravimetrically. The two samples for difference absorption spectrophotometry such as native and denatured proteins in solution were placed in reference and sample compartments, respectively, of a Shimadzu recording spectrophotometer model UV-200 of the dynode-feedback type with a fixed slit width (2 nm). The electric signal of the difference spectrum of sample-minus-reference in absorbance was memorized on two separate parallel channels in a cassette data recorder (Yasec, model 110) with a small wavelength shift (2 nm) as described previously [7 ]. The difference between the o u t p u t signals from these channels, which may be regarded as the first derivative of the difference spectrum, was recorded on a chart.

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Curve A in Fig. 1 is the difference spectrum of 4.62 mM phenylalanine dissolved in water minus phenylalanine at the same concentration in 60% dimethylsulfoxide. This spectrum indicates minima at 2 5 4 , 2 6 1 , 2 6 6 and 269 nm and maxima at 251,257, 263 and 268 nm. The difference-derivative spectrum measured directly with these solutions is shown by curve B which indicates maxima at 250, 256,262, 267 and 271 nm and minima at 253, 258, 265 and 268 nm. Yanari and Bovey [8] observed similar blue shifts of bands for benzene when the solvent was changed from hydrocarbon to water. The difference spectrum of 4.62 mM phenylalanine with 5.6 M guanidine hydrochloride minus the same phenylalanine solution without guanidine is shown by curve A in Fig. 2, and its derivative spectrum is shown by curve B. The derivative spectrum shows maxima at 252, 2 5 8 , 2 6 4 and 268 n m and minima at 255, 2 6 1 , 2 6 6 and 270 nm, which are due to the red shifts of bands. Changes of phenylalanine residues in proteins induced by denaturation with guanidine hydrochloride were next examined. Curve A in Fig. 3 is the difference spectrum of insulin with 4 M guanidine hydrochloride minus the same protein solution without guanidine, and curve B shows its derivative spectrum, which indicates maxima at 250, 256,262, 267, 271 and 281 n m and minima at 252, 258, 264, 269, 277 and 284 nm. The two peaks above 270 nm on curve B arise from changes of tyrosine bands which can be observed by common difference spectrophotometry [9]. The maxima and minima below 270 nm +0.2

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Fig. 2. Effect of guanidine hydzochloride on the s p e c t r u m of p h e n y l a l a n i n e ; the difference s pe c t rum (curve A) a n d t h e difference-derivative Spectrum (curve B) of 4.26 mM p h e n y l a l a n i n e w i t h 5.6 M g u a n i d i n e h y d r o c h l o r i d e (DH 7) m i n u s p h e n y l a l a n i n e at the same c o n c e n t r a t i o n w i t h o u t guanidine in 0.01 M Tri~ buffer (pH 7). F'~. 3. Effect of ~am~idine h y ~ o c b l o r i d e o n the spect~am of insulin; the difference s p e c t r u m (curve A) a ~ l the d i f f e ~ n e e - d e t i v a t l v e Jpeclz~m ( e u ~ B) of 174 #M insulin w i t h 4 M guanidine h y d z o c h l o r i d e (pH 7.7) m i n u s immlin at t h e smme e o n c e n t m f ~ i o n w i t h o u t guanidine in 0.075 M Tris buffe r (pH 7.7).

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agree in position and in sign with those in the difference-derivative spectrum (curve B in Fig. 1) obtained for phenylalanine in water minus phenylalanine in 60% dimethylsulfoxide, but are opposite in sign to the difference-derivative spectrum (curve B in Fig. 2) for phenylalanine in the presence of guanidine hydrochloride minus phenylalanine in its absence. The blue shift of bands thus observed indicates that guanidine hydrochloride at the concentration of 4 M denatures the insulin molecule, resulting in exposure of the phenylalanine residues to aqueous environment. Similar blue shifts of phenylalanine bands were observed when lysozyme was denatured with guanidine hydrochloride. The curves in Fig. 4 show the difference-derivative spectra obtained for lysozyme with guanidine hydrochloride at three different concentrations versus lysozyme at the same concentration in 0.05 M Tris buffer (pH 7). The spectrum changed abruptly above 4 M guanidine hydrochloride, as seen from the change from curve A to B. Curve B obtained with 4 M guanidine shows maxima at 255--274, 263 and 270 nm and minima at 260, 267 and 272--274 nm below 275 nm, which may be interpreted as blue shifts of phenylalanine bands by denaturation. Such abrupt changes above 3.5 M have been found by Hamaguchi and Kurono [ 10] who observed the changes of tryptophan band, viscosity and optical rotation of lysozyme as a function of guanidine concentration. It is well known that collagen is converted to gelatine by heat or salts [11] Curves A and B in Fig. 5 are the difference and its derivative spectra, respectively, of gelatin minus-collagen in solution. The derivative spectrum is similar i

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Fig. 4. T h e d i f f e r e n c e - d e r i v a t i v e s p e c t r a of 24.5 #M lysozyme with guan/dine h y d r o c h l o r i d e a t t h r e e d i f f e r e n t c o n e e n t r s t i o n s minus lysozyme at t h e s a m e c o n c c n t r a t / o n i n 0 , 0 5 M T r t s b u f f e r (pH 7.7); guanidine h y d r o e h l o r i d e c o n c e n t r a t i o n waS 3, 4 and 5 M for curves A, B and C, respectively. Fig. 5. T h e d i f f e r e n c e s p e c t r u m (curve A) and t h e d i f f e r e n c e - d e r i v a t i v e s p e c t r u m (curve B) of gelatin minus collagen b o t h a t 10 #M in solution (pH 4.8, ionic strength 0.06).

657 in shape to curve B in Figs 1 and 3, and shows sharp maxima at 257, 263 and 268 nm and minima at 259, 265 and 270--271 nm. It is inferred from this spectrum that some of the total 18 phenylalanine residues in the collagen molecule are exposed to aqueous environment by the heat denaturation. It was demonstrated above that difference-derivative absorbance spectrophotometry is a useful technique to investigate state changes of phenylalanine residues in proteins in solution. This is because the derivative of the molar extinction coefficient with respect to wavelength in the 250 nm region is appreciable and changes greatly with wavelength as pointed out in the previous study [7]. This exhibits many positive and negative spikes in the differencederivative spectrum, as demonstrated in this paper. The simple shift-memory method with a tape recorder may be useful for obtaining derivative spectra in laboratories. This study was supported by the grant for "Photosynthetic reaction centers" given by the Ministry of Education, and by the grant for "Life sciences" at the Institute of Physical and Chemical Research (Rikagaku Kenkyusho). References 1 2 3 4 5 6 7 8 9 10 11

Inoue, Y., Ogawa, T. and Shibata, K. (1973) Physiol. Plant. 29, 3 9 0 - - 3 9 5 Gallop, P.M. (1955) Arch. Biochem. Biophys. 54, 4 8 6 - - 5 0 0 Piez, K.A., Eigner, E.A. and Lewis, M.S. (1963) Bioche mi s t ry 2, 58--66 Gratzer, W.B. (1970) in H a n d b o o k of B i o c h e m i s t r y : S e l e c t e d D a t a for B i oc he mi s t ry (Sober, H.A., ed.), pp. B74--77, Chemical R u b b e r Co., Cleveland Well, L., Seiblis, T.S. and Herskovits, T.T. (1965) Arch. Biochem. Biophys. 111, 3 0 8 - - 3 2 0 Fromag eot, C. and Schnek, G. (1950) Biochim. Biophys. A c t a 6, 113--122 Matsushima, A., Inoue, Y. and Shibata, K..(1974) Anal. Biochem. in t h e press Yanari, S. and Bovey, F.A. (1960) J. Biol. Chem. 235, 2818--2826 Inada, Y. (1961) J. Biochem. (Tokyo) 49, 2 1 7 - - 2 2 5 Hamaguchi, K. and Kurono, A. (1963) J. Biochem. (To kyo) 54, 111--122 Von Hillel, P.H. and Wong, K-Y. (1963) Biochemistry 2, 1 3 8 7 - - 1 3 9 8