Spectroscopic Studies on the Configurational Structures of Ribonuclease T1

522 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

35567

SPECTROSCOPIC STUDIES ON THE CONFIGURATIONAL STRUCTURES OF RIBONUCLEASE T 1

YUKIO YAMAMOTO AND JIRO TANAKA Department of Chemistry, Faculty of Science, Nagoya University, Chikusa, Nagoya (Japan) (Received November 1oth, 1969) (Revised manuscript received February 6th, 1970)

SUMMARY

Ultraviolet absorption, circular dichroism, optical rotatory dispersion and fluorescence spectra of ribonuclease T 1 have been measured under various conditions. Most of the tyrosine residues and a tryptophan residue were buried in the interior of the molecule, presumably in the hydrophobic region, since the dissociation of tyrosine hydroxyl groups and the red shift oftryptophan emission band were observed with a change ofthe secondary structure, namely, to the denatured state. The a-helical content is estimated to be about 33%, the ~-structure about 24% and the random coil about 43%. The configurational structure of the molecule is discussed on the basis of the experimental informations.

INTRODUCTION

The ultraviolet absorption spectra of protein molecules are characterized by chromophores of aromatic amino acid residues and peptide groups. Phenylalanine, tyrosine and tryptophan residues absorb in the 30o-r8o-nm, and peptide groups in the zzo-r8o-nm, region. These absorption bands show sensitive changes of band positions and intensities with conformational changes of the structure and ionization of the substituent groups. The circular dichroism (CD) and the optical rotatory dispersion (ORD) are mostly used in estimating the content of a-helical structure in polypeptides and in finding the transformation of the secondary structures 1 - 4 . The fluorescence spectra are also useful in elucidating the particular details of protein, since the position and shape of the emission bands of tyrosine and tryptophan residues are greatly influenced by the ionization and the environmental effect 5 , 6 . We have investigated the configurational structures of ribonuclease T 1 by the combination of several spectral methods. The enzyme, ribonuclease Tv is specific for 3' -guanylic acid 7 ; therefore it will be of great interest to compare the details of the structure with pancreatic ribonuclease which is active for pyrimidine nucleotides. Abbreviations: CD, circular dichroism; ORD, optical rotatory dispersion.

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CONFIGURATION OF RIBONUCLEASE T 1

523

EXPERIMENTAL

Materials The sample of ribonuclease T 1 was given by the Sankyo Pharmaceutical Co. The primary structure of ribonuclease T 1 was investigated by EGAMI et al,7 and by TAKAHASHI 8. It is composed of I04 amino acids, the molecular weight being II ooo, and contains 4 phenylalanine, 9 tyrosine and I tryptophan residues. Methods The absorption spectra were obtained in a Zeiss PMQ II spectrophotometer by flushing the apparatus with nitrogen gas particularly for measurement in the shorter ultraviolet region. The circular dichroism (CD) and optical rotatory dispersion (ORD) spectra were recorded by the use of a JASC0-5 spectrophotometer, and a standard cell of IO mm path-length was used for the 24o-6oo-nm region. A thin cell of o.I-mm path length was employed for the shorter ultraviolet absorption, CD and ORD measurements. The fluorescence spectra were obtained in a Hitachi MPF-2A spectrafluorometer equipped with an adjustable thermostat. RESULTS AND DISCUSSION

Ultraviolet absorption spectra The near ultraviolet absorption spectra of tyrosine and ribonuclease T 1 in different pH solutions are presented in Fig. r. The absorption in this region is due to aromatic amino acid residues; the molar extinction coefficients at absorption maxima for these residues and the contributions to the absorbance at 276 nm are shown in Table I. The absorbance of the phenylalanine group in this region is so small that it is neglected in further discussion. The absorption spectra of tyrosine shown in Fig. I exhibit isosbestic points at 278 and 267 nm, and the change of spectrum with pH of the solution shows that a normal equilibrium is established between the hydroxyl group and water. The absorption spectra of ribonuclease Tv on the other hand, show an abrupt change in the pH range I0.3-IO.g; therefore the secondary structure of EX10"' M

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2.51-

Ail

21 I

I

11

~~

(

l '\

::~

\1

I 0.5

270

290

310

X(nm)

Fig. I. Ultraviolet absorption spectra of tyrosine as a function of pH. A. I, pH 7· I; 2, pH 8.6; 3, pH 9.0; 4, pH ro.S; 5, pH I I.H; 6, pH I2+ B. Absorption spectra of ribonuclease T 1 in the near ultraviolet region. I, pH 7· r; 2, pH g.o; 3, pH ro.J; 4, pH 10.8; 5, pH 13.5.

Biochim. Biophys. Acta, 207 (1970) 522-531

Y. YAMAMOTO, ].TANAKA

524 TABLE I ABSORBANCE OF AROMATIC AMINO ACIDS IN RIBONUCLEASE

Aromatic amino acid

Absorption maximum (nm)

Molar extinction coefficient

Phe Tyr Trp

257 275 278

I340

I9

T1

AT 276 nm

Number of aromatic amino acids

%of absorbance

4 9

0-4 68.3

5500

3!.3

the molecule will be rapidly destroyed in this pH range and most of the tyrosine groups are exposed to the solvent after the denaturation. Although no isosbestic point had been found with ribonuclease Tv two main peaks were observed at 292 and 246 nm in a strongly alkaline solution. The spectrophotometric pKA value was determined as ro.s by measuring the intensities of these two peaks. This value was not unusually large as compared with that of free tyrosine (pKA = ro.r). Although the pKA determined would be an average value for nine tyrosine residues, the spectra showed that most of the hydroxyl protons were not freely exchangeable with the solvent in acidic and neutral media below pH ro.3. However, it was not possible to determine how many tyrosine groups were exposed to the solvent in the native state. The denaturation took place at above pH rr.o, and in the intermediate pH range the secondary structure was transformed gradually with passage of time. Reversibility was tested by back titration from pH 12.0. The back titration curve did not coincide with the forward curve, showing that an irreversible denaturation of the protein had occurred at pH r2.0. These results show that most of the tyrosine groups are buried in the interior of the molecule. The far ultraviolet absorption spectra in the r85-240-nm range is also important for structural studies. In the r85-rgs-nm region the contribution of peptide chromophores will be most important (Fig. 2). A hypochromicity in the far ultraviolet spectra of peptides by a helix-coil transformation has been established9 , and the molar extinction coefficients for a single peptide group are 4300 and 6g5o (ref. ro) at rgo nm for a-helical and random coil structures, respectively. The spectra of ribonuclease T 1 showed a marked change of the intensity with the pH of the solution because the ordered structure having the a-helical conformation was transformed into the random structure by alkaline denaturation. However, the spectra of tyrosine residues would also be changed by ionization. We therefore examined the ionization effect more closely. The molar extinctions of chromophoric amino acid side chains at rgo nm are shown with the number of amino acids in ribonuclease T 1 : phenylalanine, 53 ooo (X 4); tyrosine, 38 130 (X g); tyrosine-, 47 000; tryptophan, 17 JOO (X r); histidine, 5600 (X 3); arginine, r2 450 (X r); amide, 56oo (X r2); and (cystine) 2 , 5020 (X 2) (ref. ro). The intensities of all the peptide groups at rgo nm were estimated after the contribution of chromophoric amino acid side chains had been subtracted, and the molar extinction coefficient per single peptide was found to be 6ooo at pH J.I. The value showed that the a-helical content of ribonuclease T 1 is 36% by the authentic molar absorptivities for a-helical and random structures. The estimated errors might be ± 5%. At pH ro.8 the molar extinction coefficient was increased to 66oo, and the helical content Biochim. Biophys. Acta,

207 (I97o) 522-531

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T1

525

800 ·

Fig. 2. Absorption spectra of ribonuclease T 1 in the shorter ultraviolet region. Maxima are mainly due to peptide groups. - - - , pH 7.1; - ·-, pH 10.3; ------, pH 10.9. Fig. 3· Circular dichroism of ribonuclease T 1 due to aromatic amino acid residues at different pH. - - - , pH7.1; ------, pHg.g; _ .. _. pHu.5; ······, pH13.1. Ellipticity,[ @], in degrees ·em_, · decimole-1.

was estimated to be still 13%, which suggests that short helices might be partly maintained even in this pH range. CD and ORD spectra The CD spectrum in the 240-320-nm region exhibited peaks due to the absorptions oftyrosine and tryptophan residues (Fig. 3). In the native protein, positive peaks were found at 285, 255 and 240 nm, and a small negative one at 270 nm. The positive peak at 285 nm and the negative one at 270 nm are correlated with the excitoncoupled bands of tryptopha n and tyrosine. By increasing the pH of the solution the CD spectra were changed, and at pH 9·9 a small hump was observed at 298 nm, which might be due to normally ionized tyrosine. At pH II.S several peaks were observed: a positive tyrosine peak at 298 nm, small negative humps at 287 nm, another positive one at 270 nm and a stronger negative one below 260 nm. This complex pattern might be due to tyrosine and tryptophan residues in the intermedia te of the conformat ional changes. The CD spectra were completely transforme d at pH 13.1, which showed that the secondary structure was drastically changed. Even at this pH two CD peaks were observed at 304 and 286 nm, which showed that the excition-ty pe coupling between ionized tyrosine and tryptopha n is still important. A similar interaction was found by fluorescence studies discussed later. The amino acid sequence of ribonuclease T 1 (ref. 8) shows that tyrosines and a tryptophan are close to each other at 56-59, and these groups may give rise to CD in this region. The positive peaks found at 254 and 240 nm might be assigned to the second bands of tyrosine and tryptopha n influenced by the peptide chromophores. The ORD curves in the 320-240-n m range are shown in Fig. 4· Although the location of the peaks and troughs would be modified by the existence of the stronger negative peak situated at the shorter ultraviolet region, the inflection points were found at 285 and 268 nm. These positions were in good agreement with the observed Biochim. Biophys. Acta, 207 (1970) 522-531

Y. YAMAMOTO, ].TANAKA

526

CD peaks. In alkaline solution no peaks of the Cotton effect could be detected in this region, because the tail of the stronger negative band was overlapped. The CD spectra in the shorter ultraviolet region is presented in Fig. 5: conspicuous negative peaks were found at 214 and 203 nm, a typical pattern for a right-handed a-helix. However, the maxima of the CD peaks were shifted to the shorter wavelength as compared with the usual a-helix, which were assigned at 222 and 206 nm (ref. rr). In a recent communication, ScHELLMAN AND LowE 12 report that a similar blue shift was observed on ribonuclease, and their model studies have placed the n-:rc* peak at about 212-214 nm in solvents such as water and methanol. This is the well-known blue shift of the n-:rc* band in polar solvents; and they attributed the blue shift in ribonuclease to a short or incomplete a-helix. The reason why the n-:rc* peak at 206 nm was shifted towards the blue by 3 nm is not obvious, but it has been reported that a 8-casein peak was shifted as much as 5 nm 13 . It is suggested that the mixing of ahelical, fJ and disordered structures will shift the n-:rc* band into the shorter wavelength regwn.

•4

70-1.0

/

n

~ -2.0

-3.0

/

I

I

260 280 l.(nm)

,/

I

200

210

220

230

240

200

210

220 A(nm)

230

240

/./

./

7

·4

"


300

320

Fig. 4· Optical rotatory dispersion of ribonuclease T, at different pHs.-~-, pH 7.r;----- -, pH g.o; -·-,pH ro.3; -··-,pH ro.S. [m'] =molar rotation. Fig. 5· Circular dichroism of native ribonuclease T, in water at pH 7.r3. Ellipticity, [6], in degrees· em -• · decimole- 1 •

The ORD spectra in the shorter ultraviolet region are shown in Fig. 6. The curve in neutral solution shows the typical behaviour of the a-helical structure, except that the maximum of the first trough is located at 220 nm (and is shifted towards the blue by about 13 nm) as compared with that of other helical polypeptides which is at 233 nm (ref. 14). Several methods have been proposed to estimate the helical content of polypeptides and proteins by ORD and CD spectra15 - 17 • One is by Moffit-Yang plot, and another is the trough amplitude method of the ORD curve. Actually the amplitude at 220 nm was used instead of the standard wavelength at 233 nm. The CD peak at the shorter ultraviolet region is also recommended assuming that the contribution of other conformations is not important. The values of the helical content estimated by these methods were about 33% as shown in Table II; the results were nearly in agreeBiochim. Biophys. Acta, 207 (rg7o) 522-53r

CONFIGURATION OF RIBONUCLEASE

T1

527

12

240

~

'o

-8 -12

-16

~

t'

I ,

I

A (nml

Fig. 6. Optical rotatory dispersion of native ribonuclease T 1 in water at pH 7.13 (upper curve) and denatured ribonuclease T 1 in alkaline solution at pH 13.15 (lower curve). [m'] = molar rotation.

ment with the value obtained from the hypochromicity of far ultraviolet spectra. Although it might be difficult accurately to estimate the contribution ofthe (3 structure from the present results, a method has been proposed by GREENFIELD et al. 18 by comparing the ORD curve with the constructed curve. The molecule of ribonuclease T 1 showed a blue shift of helical peaks of about 13 nm; a calculated curve was therefore constructed by shifting the a-helix curve by 13 nm towards the blue. The (3-content was estimated to be about 24% and the random coil segment was then 43%. These values may have errors of± 5%. The curve in alkaline solution showed the normal pattern of random coil structure; the magnitude of the rotation was reasonably explained by the construction curve of go% random coil and ro% a-helix. The negative tail is extended to the 230-240-nm region; the tail is somewhat shifted towards the red. Fluorescence spectra The fluorescence spectra of ribonuclease T 1 is shown in Fig. J, in which the spectral changes caused by the pH of the solution are illustrated. In the pH range TABLE II ESTIMATED CONTENT OF a-HELIX OF RIBONUCLEASE

T1

-------- ·---·-------·-------------------J'viethod

wo% helix

Ribonuclease T 1

Moffit-Yang method by a 0 Moffit-Yang method by b 0 ).c of Drude equation (nm) Molar rotation at 233 urn, [m'J Molar ellipticity at 208 urn, [(0]

l65o -540 zi\6 -rs ooo (clegrees·cm 2 ·decimole- 1) -35 ooo (degrees· cm 2 • clecimole- 1 )

-368 -200

254 -4000* - rz ooo**

39% 37% 3D% 25~~

34%

* This value was obtained at 220 nm. ** This value was obtained at 203 nm.

Biochim. Biophys. Acta, 207 (1970) 522-531

528

Y. YAMAMOTO, J.TANAKA

10

10

.fc

"c ::

8

6

-~

.D

~

280

300

320

340

360

380

400

420

7\(nml

Fig. 7· Fluorescence spectra of ribonuclease T 1 as a function of pH. - - - , pH J.I; ------. pH g.o; -·-,pH 10.3; -··-,pH Io.S; ······,pH 13.5. Fig. 8. Effect of conformational changes on the fluorescence spectra. - - - , native ribonuclease T 1 in buffer solution at pH 7.2;----- -, ribonuclease T 1 in buffer solution at pH 10.8; ribonuclease T 1 in 9 M urea solution.

J.o-ro.5, where the original conformation of the native protein would be maintained, the maximum of the fluorescence peak was observed at 320 nm. The emission was considered to arise only from the tryptophan residue because the same spectrum was observed even by exciting the tyrosine groups. This showed that the energy transfer from tyrosine to tryptophan residues is very efficient. This point of view is in accord with a recent result by LoNGWORTH19 . The fluorescence spectrum was greatly changed at pH ro.8; the two peaks were observed at 305 and 350 nm. The absorption and CD measurements, which were parallel to the fluorescence results, showed that the conformation was significantly modified at this pH. The shape of the 350-nm band was very close of that of free tryptophan in water; therefore it might be regarded as due to the tryptophan residue exposed to the environmental media by the conformational changes. The peak at 305 nm was taken to be due to tyrosine residues that were not ionized. In the mixture of water and acetonitrile (r :r, vjv) at pH ro.J, these peaks were observed at 305 and 345 nm. The dependence of the fluorescence maximum on the solvent showed that it arose from tryptophan in contact with the solvent. It is noteworthy that a complete energy transfer did not occur in the partly disordered structure. In the pH range where all the tyrosine residues in the protein would be ionized, the emission band was very close to that of ionized tyrosine. The energy level of ionized tyrosine is lower than that of tryptophan in water, so that the energy transfer will take place from tryptophan to the ionized tyrosine residues. The tryptophan emission peak observed at 320 nm in the native ribonuclease T 1 is interesting. The emission peak is unusual in being located at a shorter wavelength. TEALE 20 had shown that the fluorescence maxima of tryptophan are correlated with the dielectric constant of the solvent. The excited states of indole and tryptophan in the 300 nm region consist of two distinct states; one is the 1Lb state located at 287 nm Biochim. Biophys. Acta, 207 (1970) 522-531

CONFIGURATION OF RIBONUCLEASE T 1

529

with a weak intensity and the other is the 1 La state situated at slightly higher energy with a medium intensity 20 •21 . In the non-polar media the 1 Lb excited state is lower in energy than the 1La state, but in the polar solvent the 1 La state is much more stabilized by the solvent than is the 1 Lb state 22 and consequently the emission is observed from the 1 La level. Although WALKER et al. 23 and LONGWORTH 19 had explained the shift in terms of exciplex formation, a recent result by EISINGER AND NAVON 24 confirmed that the stabilization due to the reorientation of several solvent molecules in the solvent shell of the excited tryptophan molecule is the true origin of the red shift. Serious drawbacks to the exciplex theory 23 might be as follows. (r) Exciplex formations are generally expected between good donors and strong acceptors, but water and alcohols, which show strong solvent effect, are not recognized as good n-donors. (2) The temperature effect on the shift of the emission is rather unusual for the exciplex formation, since the excimer bands are generally increased and red-shifted down to J7°K but hindered sometimes at 4.2°K (ref. 25). (3) The position of the emission band is apparently dependent on the composition of the solvent. On these grounds the tryptophan residue of ribonuclease T 1 is considered to be buried in the hydrophobic region, where the native protein holds the portion of low dielectric constants, and the solvent molecules rotate with difficulty around the excited tryptophan residue. These conclusions are in good agreement with the conformational changes observed by ultraviolet, CD and ORD spectra. In order to show more clearly the environmental effect on the emission of the tryptophan residue, the spectra in the native state were compared with that of denatured ribonuclease T 1 . The fluorescence spectrum of ribonuclease T 1 denatured by 9 M urea resembled that at pH ro.8; hence it was confirmed that the tryptophan residue is exposed to the surrounding media in this condition. The emission intensity is three times stronger in the native state than in the denatured state. The quenching of the fluorescence by the motion of the solvent molecule was suggested by EISINGER AND NAVON 2 i from the effect of deuterium. Next, we studied the effect on the fluorescence spectrum 26 of changing the temperature from 5 to 65°. Two wavelengths for excitation were set at 278 and 295 nm, because both tyrosine and tryptophan residues are excited at 278 nm but only the tryptophan residue is excited at 295 nm. In the temperature range 5-50°, the fluorescence spectra had maxima at 320 nm due to the tryptophan residue irrespective of the excitation wavelengths, which showed that no structural change had occurred.

Fig. g. Effect of heat denaturation on the fluorescence spectra; for the excitation at 295 nm (A) and 278 nm (B). respectively. Temperatures: I. so; 2. IS 0 ; 3. 30°; 4. 40°; s. sao; 6. SS 7. 60°; 8. 650· 0

;

Biochim. Biophys. Acta, 207 (I970) 522-53I

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Y. YAMAMOTO, J. TANAKA

Heating of the solution led to the reduction of the fluorescence intensity, as shown in Fig. g. At 55°, the fluorescence intensity was decreased markedly, and the excitation at wavelengths 278 and 295 nm produced different band shapes. In the spectrum for 278 nm excitation the fluorescence band originating from tyrosine was found besides the tryptophan band, whereas for 295 nm excitation the fluorescence maximum was observed at 333 nm, which corresponds to the tryptophan band in the environment of the medium polarity. This shows that the energy transfer from tyrosine to tryptophan residues was hindered to some extent, because the secondary and tertiary structures of the protein were changed. In the temperature range above 55°, the fluorescence spectrum for 278 nm excitation showed two maxima at 305 and 350 nm, whereas that for 295 nm excitation had only one maximum at 350 nm. On cooling the solution after the attainment of 65°, we observed the same fluorescence spectra as that seen on heating it. These results tell us that the full denaturation occurs above 65°. In conclusion, the combined spectral measurements on ribonuclease T 1 reveal that: (r) most of the tyrosine residues, which are centred perhaps in the sequences (38)-(68), may be buried in the interior of the protein molecule and may be involved in the hydrophobic interaction; (2) the tryptophan residue (59) is particularly shielded from the solvent molecules in the native state; (3) the a-helix content is about 33%, the /J-structure about 24% and the random coil about 43%; (4) the a-helix may consists of rather short or incomplete helices; (5) the conformational changes are reversible in the pH range 7.r-ro.s and in the temperature range 5-65°. These results may be relevant to the mode of the enzyme action and the structure of the native ribonuclease T 1 . ACKNOWLEDGEMENTS

The authors are grateful to the Ministry of Education for a grant, and to the Sankyo Pharmaceutical Co. for the gift of samples. They also thank Professor F. Egami of Tokyo University for much helpful discussion. REFERENCES I 2 3 4 5 6 7 8 9 ro II I2 I3 14 IS 16 17

J. J.

T. YANG AND J. F. FosTER,]. Am. Chem. Soc., 76 (I954) I588. T. YANG AND P. DoTY,]. Am. Chem. Soc., 79 (I957) 761. P. URNES AND P. DoTY, Advan. Protein Chem., I6 (Ig6I) 401. C. ScHELLMAN AND ]. A. SCHELLMAN, Compt. Rend. Lab. Carlsberg, Stir. Chim., 30 (I958) 463. S. V. KoNEV, Dokl. Akad. Nauk SSSR, u6 (I957) 594· R. F. STEINER AND H. EDELHOCK, Nature, I92 (Ig6r) 873. F. EGAMI, K. TAKAHASHI AND T. UcHIDA, Progress in Nucleic Acid Research and Molecular Biology, Vol. 3, Academic Press, New York, I964, p. 59· K. TAKAHASHI,]. Biol. Chem., 240 (I965) PC 4u7. K. IMAHORI AND]. TANAKA, j. Mol. Biol., I (I959) 359· W. B. GRATZER, in G. D. FASMAN, Poly-a-Amino Acids, Marcel Dekker, New York, I967, p. I77-23S. G. HoLZWORTH, W. B. GRATZER AND P. DoTY,]. Am. Chem. Soc., 84 (I962) 3I94· J. A. ScHELLMAN AND M. J. LowE,]. Am. Chem. Soc., 90 (I968) I070. S. N. TIMASHEFF, H. Susui, R. TowNEND, L. STEVENS, M. ]. GoRBUNOFF AND T. F. KuMoSINSKI, Conformation of Biopolymers, Vol. I, Academic Press, New York, I967, p. I73· E. R. BLOUT, I. ScHMIER AND N. S. SIMMONS,]. Am. Chem. Soc., 84 (I962) 3I93· N. S. SIMMONS, C. CoHEN, S. G. SzENT-GYORGYI, D. B. WETLAUFER AND E. R. BLoUT, ]. Am. Chem. Soc., 83 (I961) 4766. H. HASHIZUME, l\1. SHIRAKI AND K. IMAHORI, ]. Biochem., 62 (1967) 543· J. T. YANG AND P. DoTY,]. Am. Chem. Soc., 79 (1957) 761.

Biochim. Biophys. Acta, 207 (I97o) 522-531

CONFIGURATION OF RIBONUCLEASE

r8 I9 20 2! 22 23 24 25 26

T1

53 I

N. GREENFIELD, B. DAVIDSON AND G. D. FASMAN, Biochemistry, 6 (1967) 1630. J. W. LONGWORTH, Photochem. Photobiol., 7 (1968) 587. F. W. J. TEALE, Biochem. ]., 76 (1960) 381. E. A. CHERNITSKII AND s. V. KONEV, Dokl. Akad. Nauk SSSR, 8 (r964) 2j8. N. MATAGA, Y. ToRIHASHI AND K. EZUMI, Theoret. Chim. Acta, 2 (1964) Ij8. M.S. WALKER, T. W. BEDNAR AND R. LUMRY, j. Chem. Phys., 47 (r967) !020. J. EISINGER AND G. NAVON, j. Chem. Phys., 50 (1969) 2069. J. TANAKA, Bull. Chem. Soc. japan, 36 (1963) 1237. S. V. KoNEV, Fluorescence and Phosphorescence of Proteins and Nucleic Acids, Plenum Press, New York, 1967, p. 83.

Biochim. Biophys. Acta, 207 (1970) 522-531