Circular dichroism and Cotton effects in the near-ultraviolet spectral region of aldolase

Circular dichroism and Cotton effects in the near-ultraviolet spectral region of aldolase

366 SHORT COMMUNICATIONS BBA 33 007 Circular dichroism and Cotton effects in the near-ultraviolet spectral region of aldolase There have been an in...

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366

SHORT COMMUNICATIONS

BBA 33 007

Circular dichroism and Cotton effects in the near-ultraviolet spectral region of aldolase There have been an increasing number of reports describing Cotton effects and dichroic bands associated with optical activity in the aromatic side chains of proteins 1-3. The existence of such effects provides a potentially useful approach for investigating the conformation of the side chain on the protein and in some cases the optical activity of the chromophore has been correlated with its location in an asymmetric environment in the interior of the molecule~, ~. We wish to report the occurrence in the near-ultraviolet spectral region of dichroic bands and the corresponding Cotton effect in aldolase. Crystalline, rabbit muscle aldolase (fructose-i,6-diphosphate D-glyceraldehyde3-phosphate-lyase, EC 4.1.2.13) was purchased from C. F. Boehringer and Soehne, Mannheim, as a crystallised suspension in 2 M a m m o n i u m sulphate. Before use, this solution was dialysed at 4 ° against o.oi M NaC1. In this solvent s°20 ~ 7.2 S. The protein concentration was determined from the absorbance at 280 m/z using a value of E~ ~2 ~ 9 -1 (see ref. 4). E n z y m e assays were carried out b y the method of JAGANNATHAN, SINGH AND DAMODARAN 5.

Optical r o t a t o r y dispersion (ORD) curves were recorded on a Bendix-Ericson "Polarmatic 62" recording spectropolarimeter. Dispersion curves were recorded at least twice in a silica cell, I-cm path length, at 25 °. Duplicates agreed within the noise level of the signal. All rotations were proportional to the protein concentration. The signal-to-noise ratio was greater than 20 in the range 500 to 300 m/z and decreased to about 15 at 23o m/z. Entrance and exit slit widths were o, 5 and 0.4 m m respectively in the range 500 to 30o m/z and 1. 5 and 1.2 m m in the range 300 to 230 m/z. The magnitude of the rotation was independent of slit width in this range. Artifacts of the type reported by RESNICK AND YAMAOKA6 were found to be negligible under the conditions of measurement. Circular dichroic spectra were recorded at room temperature in a Roussel-Jouan Dichrographe calibrated with a standard solution of epiandrosterone in methanol. The wavelength calibration was accurate to =~ I m/z. All measurements were made at the m a x i m u m sensitivity of the machine. The exit-slit width was maintained below 1. 5 mm. Spectra were recorded 3 times at concentrations between 12 and 1.2 mg/ml in quartz cells of I.O-, o.5- and o.i-cm path length. Rotational strengths were estim a t e d from the areas of the dichroic bands b y tracing the spectra onto thick card, which was then weighed. The estimated error is =[= 50 %. The circular dichroic spectrum of aldolase in o.oi M NaC1, p H 6.0, (Fig. I) showed a negative band, centred at 295 m/z, between 3Io and 288 m/z, rotational strength, R --~ I.O. IO-4~ c.g.s, per mole of average amino acid residue assuming 115 as the mean residue molecular weight; a positive band centred at 280 m/z, between 288 and 268 m/z, R ~ I.o. IO-~2 c.g.s, per mole of amino acid residue. Adjacent to this latter band was the shoulder of a negative band with a m i n i m u m at about 220 m/z. The rotational strength of this b a n d was not estimated. At p H 2.5 where aldolase is Abbreviation : ORD, optical rotatory dispersion.

Biochim. Biophys. Acta, 133 (1967) 366-369

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dissociated into threC, s or four 9 unfolded subunits, the two dichroic bands centred at 295 and 280 m/~ disappeared and the band at 220 m/~ decreased in magnitude and in band width (see dotted line, Fig. I). The ORD curve of native aldolase in o.oi M NaC1, p H 6.0, showed negative rotations over the range 5oo to 230 m/~ with a negative Cotton effect at about 235 m/,. In addition an anomaly was observed at 290 m/~ (Fig. 2). The shape and position of the anomaly can be accounted for in terms of the two overlapping Cotton effects which are associated with the two adjacent dichroic bands between 31o and 268 m/~.

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Fig. i. (a) C i r c u l a r d i c h r o i c s p e c t r u m of a l d o l a s e in o . o i M NaC1. - - , p H 6.0; . . . . . . , p H 2.5. P r o t e i n c o n c e n t r a t i o n : 2. 5 m g / m l , i n i - c m p a t h - l e n g t h cell. T h e c u r v e s a r e c o p i e s of t r a c i n g s , a c t u a l size, r e c o r d e d o n t h e D i c h r o g r a p h e . - . . . . , a b s o r p t i o n s p e c t r u m of a l d o l a s e . (b) C i r c u l a r d i c h r o i c s p e c t r u m of a l d o l a s e in o . o i M NaC1. - - , p H 6.0; . . . . . . , p H 2.5. P r o t e i n c o n c e n t r a t i o n : 1.25 m g / m l , i n o . i - c m p a t h - l e n g t h cell. T h e c u r v e s a r e c o p i e s of t r a c i n g s , a c t u a l size, r e c o r d e d o n t h e D i c h r o g r a p h e . zJe is t h e d i f f e r e n c e in a b s o r p t i o n coefficient b e t w e e n l e f t - a n d r i g h t - c i r c u l a r l y p o l a r i s e d l i g h t p e r m o l e of a m i n o a c i d r e s i d u e t a k i n g t h e m e a n r e s i d u e m o l e c u l a r w e i g h t a s 115 . F i g . 2. O R D c u r v e of a l d o l a s e i n o . o i M NaC1, p H 6.o, s h o w i n g (a) t h e C o t t o n effect a t 29o m/~; (b) t h e C o t t o n effect a t 235 m u. [m']~, ~ 3/(n 2 + 2)- ( M R W / I o o ) . [~1~,, w h e r e [c~]~. is t h e r o t a t i o n a t w a v e l e n g t h )4 M R W is t h e m e a n r e s i d u e m o l e c u l a r w e i g h t , t a k e n a s 115; n is t h e r e f r a c t i v e i n d e x of t h e s o l v e n t a t w a v e l e n g t h 2; n w a s t a k e n as 1.33 o v e r t h e r a n g e 230 t o 500 m # .

Two such adjacent bands of approximately equal and opposite rotational strength will give rise to a Cotton effect having two minima and one maximum, centred approximately at the point of intersection of the bands t°. This curve, superimposed on the background dispersion of the peptide bond would account for the anomaly shown in Fig. 2. The effect disappeared at p H 2.5 and thus reflected the behaviour of the dichroic bands, neither was it present in aldolase denatured in 2.5 M guanidine hydrochloride, 5.0 M urea or sodium dodecyl sulphate (I %, w/v). An analysis of the curve in the range 500 to."33 ° m/~ according to the procedure of MOFFIT AND WANG14 yielded a straight line with slope, --b0, equal to 251 and intercept, ao, equal to --IOO. The value of b0 corresponded to 4 ° % effective helical content. The helical content estimated from the rotation at the minimum at 235 m/~, according to the method of SIMMONS et al. 15, corresponded to 24 %. Aldolase which had been denatured at p H 2.5, and then renatured by adjusting the solution to p H 7.0 either by the rapid addition of alkali or by dialysis against B i o c h i m . Bi ophy s . Acta, 133 (1967) 3 6 6 - 3 6 9

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o.I M sodium phosphate bufferT,8,n, exhibited the Cotton effect at 290 m/z, despite the recovery of only IO % of the original enzymic activity. The two dichroic bands centred at 295 and 280 m/~ probably arise from optically active electronic transitions in the tryptophan and tyrosine side chains of the protein. DONOVAN12 has shown that only one of the ten or eleven tryptophan chromophores in aldolase is exposed to solvent; the rest are "buried" in the interior of the protein. Such "buried" chromophores have their spectrum displaced to longer wavelengths (the red shift) 1~,13. The coincidence of the negative dichroic band at 295 m/, and the absorption spectrum of the native protein implies that the dichroism is associated with optical activity in the buried tryptophan residues. The band centred at 28o m/z m a y arise from optical activity in tryptophan or tyrosine residues since there are electronic transitions associated with both chromophores in this region. 28 of the 42 tyrosine residues are "buried" in the native protein 12, These m a y well be in positions of hindered rotation and optical asymmetry. Denaturation of the native enzyme at low p H destroyed the optical activity of the chromophores. On renaturation their optical activity was restored. The negative dichroic band centred at about 22o m/~ must in part be due to the secondary structure of the polypeptide chain which appears to be predominantly a-helix. At p H 2.5, where the molecule is largely unfolded, the intensity and band width diminished. The latter observation suggests that the band observed at p H 6.o m a y be made up in part of contributions from chromophores other than the peptide bond. The discrepancy between the values of helical content estimated from b0 and [m'12a5 is clearly due to the presence of dichroic bands in the near-ultraviolet. The estimate based on Em']2~5 is probably more reliable. These results show that there are dichroic effects associated with the tertiary or quaternary structure of aldolase. A systematic study of the effect of p H and solvent on the dichroism m a y yield information about the secondary, tertiary and quaternary structure of the protein. We wish to thank Professor R. C. COOKSON, Southampton University, for use of the Dichrographe.

Biochemistry Department, Oxford University, Oxford (Great Britain)

I 2 3 4 5 6 7 8 9 1o i I

K. R. LEONARD I. 0. WALKER

~X~.S. SIMMONS AND E . R . BLOUT, Biophys. J . , i (196o) 55D. V. MYERS AND J. T. EDSALL, Proc. Natl. Acact. Sci. U.S., 53 (1965) 169. S. BEYCHOK, Proc. Natl. Acad. Sci. U.S., 53 (1965) 999. G. SZABOLCSI AND E . BlSZKU, Biochim. Biophys. Acta, 48 (1961) 335. V. JAGANNATHAN, K. SINGH AND M. DAMODARAN, Bioehem. J., 63 (1956) 94R . A. RESNICK AND K. YAMAOKA, Biopolymers, 4 (1966) 242. E . STELLWAGEN AND H . K. SCHACHMAN, Biochemistry, i (1962) lO56. W . C. DEAL, W . J. RUTTER AND •. E. VAN I-IOLDE, Biochemistry, 2 (1963) 246. C. TANFORD AND I{. I~A~ATAHARA,Biochemistry, 5 (1966) 1578. I. TINOCO, J. Am. Chem. Soc., 86 (1964) 297. E . W . WESTHEAD, L. BUTLER AND P. D. BOYER, Biochemistry, 2 (1963) 927.

Received September i9th, 1966 Biochim. Biophys. Acta, 133 (1967) 3 6 6 - 3 6 9

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J. W. DONOVAN, Biochemistry, 3 (1964) 67. S. YANARI AND F. A. BOVEY, J. Biol. Chem., 235 (196o) 2818. W. MOFFIT AND Y. T. YANG, Proc. Natl. Acad. Sci. U.S., 42 (1956) 596. N. S. SIMMONS, C. COHEN, A. G. SZENT-GYoRGYI, D. B. WETLAUFER AND E. t{. BLOUT, J . A m . Chem. Soc., 83 (1961) 4766.

Biochim. Biophys. Acta, 133 (1967) 366-369

BBA 33 003

Separation of dansyl-amino acids by polyamicle layer chromatography The highly fluorescent sulfonamide derivatives of dimethylaminonaphthalene5-sulfonyl chloride (dansyl chloride, DNS-C1) are favored for amino terminal analysis of peptides and proteins because they are quite stable toward acid hydrolysis and can be detected at ioo-fold greater sensitivity than fluorodinitrobenzene derivatives. Though, for identification, dansyl amino acids have been effectively separated electrophoretically, the equipment used has been both hazardous and expensive1, 2. We have found that all of the dansyl derivatives of amino acids commonly present in proteins are readily separable by thin-layer chromatography on durable s-polycaprolactam bonded to solvent-resistant polyester sheets ('polyamide layers") as prepared by WANG,WANG AND LIN 3. The system described here is rapid, simple and relatively inexpensive. Approx. 1-2 m/~moles of peptide or protein in 20/~l o.I M NaHCOs contained in a 6 mm × 15 mm borosilicate tube is dansylated by the addition of 20/~1 DNS-C1 reagent** (I mg/ml in acetone). The tube, covered with Parafilm, remains at room temperature 2-16 h; the cover is removed, and the contents are evaporated to dryness in a vacuum desiccator (water aspirator) over sulfuric acid. A 5o-~1 volume of 6 M HC1 is added, the tube is sealed and placed in a lO5 ° oven for 6-16 h for hydrolysis, then cooled and opened and its contents are evaporated to dryness over NaOH pellets. The sample is transferred with acetone-glacial acetic acid (3 : 2) to a polyamide sheet for chromatography, being deposited in a 2-mm diameter spot in a corner (2 cm from each edge). Development in a covered jar by ascending solvent flow employs: I. w a t e r - 9 o % formic acid (200:3, v/v), initial vector and, after air drying; 2. benzeneglacial acetic acid (9:1, v/v), 9 °0 vector. The solvent fronts migrate approx. IO cm in 30 and 60 min, respectively, in solvents I and 2. Other effective solvent systems include: 3. n-heptane-n-butanol-glacial acetic acid (3 : 3 : I, by vol.) ; 4. chlorohydrinpyridine-toluene: o.8 M NH4OH (3:1.5:5:3, by vol.); 5. chlorobenzene-glacial acetic acid (9:I, v/v). System 3 is more effective than 2 for separation of DNSglutamic and DNS-aspartic acids. The separated dansyl amino acids are visualized by ultraviolet irradiation (caution, wear protective glasses). The reaction by-products DNS-OH and DNS-NH 2 do not interfere. Separation of a mixture of amino acids carried through the entire procedure for peptides is illustrated in Fig. i, which also indicates the positions for the mono-substituted bifunctional amino acids tyrosine and lysine determined by A b b r e v i a t i o n : DNS-, d i l n e t h y l a m i n o n a p h t h a l e n e - 5 - s u l f o n y l (dansyl). * Cheng-Chin T r a d i n g Co. Ltd., No. 75 Sec. i, H a n k o w St., Taipei, T a i w a n . ** Pierce C h e m i c a l Co., Rockford, Ill., U.S.A.

Biochim. Biophys. Acta, 133 (1967) 369-37 °