The solvent effect on the fluorescence and light absorption of riboflavin and lumiflavin

BIOCHIMICA ET BIOPHYSICA ACTA

289

BBA 45177 T H E SOLVENT E F F E C T ON T H E F L U O R E S C E N C E AND L I G H T A B S O R P T I O N OF R I B O F L A V I N AND L U M I F L A V I N JACEK KOZIOL* AND EDUARD KNOBLOCH Central Research Institute of Food Industry, Prague, and Institute for Pharmacy and Biochemistry, Prague (Czechoslovakia)

(Received September 7th, 1964)

SUMMARY

Changes in fluorescence intensity of riboflavin and lumiflavin with the nature of the solvent were studied. A series of mixtures of ethanol, acetone and dioxane with water were used as solvents. Changes in absorption and fluorescence spectra expressed as transition energies, apparent absorption coefficients and quantum yields of fluorescence were correlated with each other, with dielectric constants and with the Z-values, expressing solvent polarity of the solvent mixtures used. Rough linearity was observed for all parameters except of dielectric constants for flavin solutions containing no more than 9 ° % of organic solvent. In riboflavin solutions containing higher concentrations of organic solvents deviations from linearity were observed. The possibility is discussed, that in such higher concentrations of organic solvents relative measurements accepted do not adequately reflect changes in flavin molecules. In solvent mixtures of lowest polarity riboflavin and, to a greater extent lumiflavin, were photodecomposed much faster than in aqueous solutions. It is suggested that the decrease of the stability of flavin molecules is caused by lowering of the degree of association with water molecules, and by a secondary solvent polarity effect on the electron system of light-excited flavin molecules.

INTRODUCTION An increase in riboflavin fluorescence intensity was observed in a range of mixtures of organic solvents with water, used for elution of riboflavin from a cation exchanger, a stage included in a fluorimetric method for riboflavin determination in food products 1. Moreover, for riboflavin solutions in these solvent mixtures the use of the differential fluorimetric method proved impossible, because, in contrast to riboflavin solutions in water, the intensity of fluorescence was found to be different at various wavelengths of exciting light S. To study this phenomenon, we tested the effect of ethanol, acetone, 1,4-dioxane, and their mixtures with water, on the fluorescence of riboflavin, and also on lumiflavin because it is more soluble in these organic solvents. The results of preliminary experiments 3 encouraged us to make a more extensive study, part of which is reported in the present paper. * Present address: Department of Food Products Quality, High School of Economics, Poznafi, Poland. Biochim. Biophys. Acta, io2 (I965) 289-300

290

J. KOZIOL, E. KNOBLOCtt

EXPERIMENTAL

Apparatus Fluorescence intensity was measured using a laboratory-constructed filter fluorimeter of high sensitivity and selectivity with right-angle illumination (lowpressure, low-intensity mercury lamp, with bulb covered with fluorescent layer, interference filters, Zeiss (Jena) photomultiplier, I cm fused quartz rectangular cuvettes) 4. Fluorescence spectra were recorded with the same apparatus with flint-glass prism monochromator VSU-I (Zeiss, Jena) instead of secondary filter. Absorption curves were taken with a Unicam Model SP-7oo recording spectrophotometer, i cm "silica" absorption cells.

Materials Riboflavin: Merck, Darmstadt. Aqueous solutions containing Ioo /~g/ml. Lumiflavin: prepared by photolysing riboflavin solution in I N NaOH, extracting with chloroform, drying in vacuum, several times recrystallizing from hot water. Chromatographically pure. Fluorescein: Lachema, Prague. Ethanol: commercial absolute, dried with magnesium and redistilled. Acetone: p.a. grade, dried with K 2CO3, refluxed with KMnO 4. Dioxane: p.a. grade, fractionated after refrigeration, mixed with sodium and distilled. Paper chromatography: W h a t m a n No. I paper. Solvent mixtures: n-butanol-acetic a c i d - w a t e r (4:1:5, v/v), 5 % Na~HPO4, circular and descending techniques.

RESULTS

Fluorescence intensity was measured from aqueous solutions of riboflavin and lumiflavin and for both flavins in a series of mixtures of IO, 25, 50, 80, 9 ° % (v/v) ethanol, acetone and dioxane with water, 95 % for riboflavin and pure solvents for lumiflavin. The concentration of flavins was 0.5/~g/ml. Measurements were carried out with the filter fluorimeter using exciting-light wavelengths of 436 m/~ and 366 m/~, secondary filter 546 m/~ (see ref. 4). Using excitation with 436-m/~ light it was found that the increase in fluorescence intensity was proportional to the increase of organic solvent concentration. The proportionality was valid in the flavin concentration range 0.5 to IO ~g/ml, and did not change on standing for 24, 49 or 72 h before measurement. The effect of fluorescence self-quenching for particular concentrations was the same for flavin solutions in water and in organic s o l v e n t - w a t e r mixtures. Excitation at 366 m/~ produced a small increase in fluorescence intensity in mixtures containing no more than 80 % of ethanol, and only 50 % of acetone or dioxane. With higher concentrations of acetone or dioxane the fluorescence intensity decreased to below the levels obtained for flavin solutions in water. Values for fluorescence concentration quenching at some flavin concentrations were different for each type of mixture series, being lower for all solvent mixtures than for aqueous solutious. For further study of the phenomenon, fluorescence spectra of riboflavin and lumiflavin were recorded in analogous series of solvent mixtures using both excitation wavelengths. Fluorescence intensity values measured at m a x i m a were proportional to those observed in the filter fluorimeter. The shape of the spectral curves was the Biochim. Biophys..~lcta, lo2 (1965) 289-300

SPECTROSCOPY

291

OF RIBOFLAVIN AND LUMIFLAVIN

same for each solvent series at 436 m/~ and at 366 m/~, and a small hypsochromic shift of the m a x i m a was observed. As the emission of fluorescence is a function of light absorption of a molecule, absorption spectra of riboflavin and lumiflavin were measured in the series of solvent mixtures used. The flavin concentration in all mixtures was io/~g/ml, with the exception of riboflavin solutions in 95 % mixtures in which the concentration was 5/~g/ml. In contrast to absorption curves for both flavins in water solution taken as comparison standards a hypsochromic shift of the m a x i m u m and a drop of absorbancy at 366 m/~ were observed in solutions of organic solvents (Fig. I). Other changes in recorded spectra were insignificant. As the position of the near-ultraviolet absorption band and absorbancy at this wavelength were remarkably sensitive to the composition of solvent mixtures, both values were used in further investigation as relative measures of solvent effect on absorption spectra. F r o m values presented in Table I, and from the shapes of absorption curves (Fig. I), it is apparent that fluorescence intensity measurements in organic s o l v e n t - w a t e r mixtures were affected strongly when the 366-m F band was used for excitation, and only slightly when the 436-m/z band was used. 4O

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F i g . I. A b s o r p t i o n s p e c t r a of f l a v i n s o l u t i o n s , l ~ l a v i n c o n c e n t r a t i o n IO # g / m l . a : R i b o f l a v i n i n water and in 90 % mixtures of ethanol, acetone and dioxane with water, b: Lumiflavin in water and in pure ethanol, acetone and dioxane. , water; ..... , ethanol, pure and mixed with water; ..... , acetone, pure and mixed with water; .... , dioxane, pure and mixed with water.

In order to determine the solvent effect on fuorescence of riboflavin and lumiflavin, fluorescence spectra of both compounds using the 436-m/z excitation band were recorded. Because changes in the fluorescence spectra are determined not only b y intensity changes at maxima, but also b y changes of position of m a x i m a and shape of curves obtained, it was desirable to use q u a n t u m yields of fluorescence (~), which were derived from the equation: q~z = FI'q~2"A2/F2"A1 (ref. 4) Biochim. Biophys. Acta, lO2 (1965) 2 8 9 - 3 o o

O ©

~D

I

IN POSITION

27 600

95*

*

27 500

90

.

o.24o

27 7oo

27 2oo

8o

too

o.261

27 15o

28 500 .

.

0.222

0.229

.

AND

.

28 600

28 300

27 7oo

27 15o

26 8oo

26 600

.

(cm-1)

3laximlon position

Dioxane

BAND

--

o.212

0.223

o.233

o.277

o.269

0.284

OF

28 700

28 35 °

28 t o o

27 6oo

27 35 °

27 200

27 I 2 5

(cm-1)

Maximum position

0.284

.

0.292

o.3oo

o.327

o.343

0.347

0.352

Absorba.ncy

RIBOFLAVIN

Lumiflavin - Ethanol

ABSORBANCIES

Absorbancy

IN

R i b o f l a v i n c o n c e n t r a t i o n 51~g/inl, a b s o r b a n c y v a l u e s m u l t i p l i e d b y 2.

0.242

0.245

o.255 28 200

o.268

26 8oo

26 85o

5o

o.27o

o.282

26 65o

25

0.276

26 650

.

0.282

.

26 55 °

.

Absorbancy

io

.

(cm- l)

Maximum position

0.283

Absorbanc_v

ABSORPTION

26 500

o (water)

(cm ~)

Percentage Riboflavin organic solvent Ethanol concentration (v/v) Maximum position .4 cetone

OF NEAR-ULTRAVIOLET

F l a v i n c o n c e n t r a t i o n I0 p g / m l .

OF SOLVI~NT M I X T U R E S

CHANGES

TABLE

. 29 500

29 ooo

28 5oo

27 8oo

27 45o

27 250

.

(cm-1) .

.

Maximum position

o.27t

o.281

o.292

o.318

o.335

0.345

.

.

IN

29 900

29 200

28 75 °

27 9oo

27 45o

27 3 ° 0

.

(cm-1)

Maximum position

Dioxane

SOLUTIONS

Absorbancy

LUMIFLAVIN

Acetone

AND

A

0.287

o.281

o.289

o.3o5

o-32o

0.344

Absorbancy

SERIES

© t~

©

©

©

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II

1

0

I00

95

I~

90

29.65

28.4

27. 4

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5°

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25

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23.6

io

23. 4

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o

(v/v)

(#-1)

1.9o9

1.9o3

I. 899

1.895

1.89

I 882

1.882

~ x lO~ Maximum position

Percentage Riboflavin organic solvent Ethanol concentration

F l a v i n c o n c e n t r a t i o n 2. 5 / t g / r n l .

36.3

32.5

3° .o

26.9

24.6

23.65

q} × lO 2

Acetone

-

-

1.918

1.915

1"912

1.899

1.897

1.882

(t* 1)

lI6raximum position × Io z

--

37.4

33.6

31"4

27.2

25.0

23.75

~

D ioxane

--

1.918

1.915

1"91°

1.899

1.892

1.882

(#-1)

Maximum position x lO 2

43.5

.

37.0

34.5

30.4

28. I

26.9

26.0

¢

Ethanol

.

Lumi[tavin

1.92o

1.918

1.915

1.91o

1.9o 3

1.899

1.899

(/~ 1)

.

Maximum position

x lO 2

61.o

.

48.0

41-9

34.2

30.3

27. 7

¢

Acetone

1.928

.

1.922

1.918

1.91o

1.9o 3

1.899

(#-~)

.

Maximum position

× Io

71.o

51.9

44.6

35.6

30.5

27. 4

~

Dioxane

1.93 °

1.922

1-915

1.91o

1.9o3

1.899

(#-1)

Maximum position

CHANGES IN QUANTUM YIELDS OF FLUORESCENCE AND IN POSITIONS OF FLUORESCENCE MAXIMA OF RIBOFLAVIN AND LUMIFLAVIN SOLUTIONS IN A SERIES OF SOLVENT MIXTURES

TABLE

Oq

~D

t~

>

O t~

0

0 &o ¢3 0

t~ C3

294

]. KOZlO/, E. KNOBLOCH

where : F = fluorescence intensity (area under emission curve) and A -----absorbancy; I : examined c o m p o u n d ; 2 : standard compound. As the m o n o c h r o m a t o r - p h o t o m u l t i p l i e r combination showed maximal sensitivity at 350-520 m/~, only short-wave halves, obtained b y dividing the recorded spectra with an ordinate at m a x i m u m , were used for area measurements. This simplification, together with the use of uncorrected fluorescence spectra, are based on the satisfactory coincidence of flavin spectra both a m o n g themselves and with the spect r u m of fluorescein in o.I N N a O H solution which was used as standard 5 (Fig. 2).

35(

30(

25(

g m

g Ix

10.(

5(

2.2

2.1 2.0 1.9 Waven u m b e r (~-1)

1.8

F~g. 2. Fluorescence emission spectra (uncorrected) of riboflavin, lumiflavin solution. Concentration 2. 5 ffg/m]. F, fluorescein solution in o.i N N a O H ; 0.o 3 IV[ p h o s p h a t e buffer (pH 7.0) ; WL, lumiflavin in 0.03 M p h o s p h a t e buffer DR, riboflavin solutions in 95 % m i x t u r e s of ethanol, acetone, a n d d i o x a n e w i t h lumiflavin in pure ethanol, acetone a n d dioxane.

a n d fluorescein in WR, riboflavin in (pH 7.0) ; ER, AR, w a t e r ; EL, AL, DL,

E m p l o y i n g this simplified method, and accepting as s t a n d a r d value a fluorescein q u a n t u m yield of fuorescence equal to 0.92, a q u a n t u m yield 0.234 for riboflavin solution in 0.03 M phosphate buffer (pH 7.0) was obtained. This value satisfactorily agrees with values of 0.25-0.26 cited in the literature6,% Q u a n t u m yield values for b o t h flavins in aqueous solutions and in solvent mixtures, and shifts of fluorescence maxima, are shown in Table U. The concentration of riboflavin, lumiflavin and fluorescein in all solutions was 2.5 ffg/ml. I n all organic solvent mixtures an increased photolability of riboflavin, and also of lumiflavin (regarded as relatively photostable in aqueous solutionsS), was Biochim. Biophys. Acta, lO2 (1965) 289-300

SPECTROSCOPY OF RIBOFLAVIN AND LUMIFLAVIN

295

observed. Riboflavin solutions in water and in 99% s o l v e n t - w a t e r mixtures and lumiflavin solutions in water and in pure solvents, r a v i n concentration 0.5 ~g/ml, were illuminated with a IOO-watt tungsten lamp at 30 cm distance. Solutions were in transparent, equal-sized, stoppered, glass tubes; fluorescence intensity changes were measured on the filter fluorimeter with exciting light 436 m/~, and secondary filter 546 m ~ (Fig. 3). r

I 150f

200

.•100

150'

~- 50 !o L

0 30

90

150 210 270 330

30

90

150 210 270 330

Time of illuminotion (rain)

Fig. 3. Photolytic decomposition of riboflavin a n d lumiflavin solution. Concentration 0. 5/~g/ml. Left figure : Riboflavin solutions in w a t e r a n d in 99 % m i x t u r e s of ethanol, acetone a n d d i o x a n e w i t h water. R i g h t figure: Lumiflavin solutions in w a t e r a n d in p u r e ethanol, acetone a n d dioxane. V~Z, E, A, D: water, ethanol, acetone a n d dioxane, respectively.

All operations with flavin-containing solutions were carried out in a darkened laboratory illuminated with weak red light. Measurements of absorption and fluorescence spectra and fluorescence intensity were made at a constant temperature of 25 °. DISCUSSION

The influence of organic solvents on flavin fluorescence intensity was first observed b y COHEN (see ref. 9) and later b y BESSEY et al. 1°, but because of the low selectivity of the fluorimeters used the data are not accurate. As an initial check, values obtained for fluorescence yields of riboflavin and lumiflavin solutions in all solvent mixture series were used to compare with absorption m a x i m a shifts. The m a x i m a were converted to transition energies b y means of the relation: ET (kcal/mole) ---- 2.859" lO-3 ~ (in cm-1) 11. When ~b-values were plotted against ET-values straight lines were obtained up to 9 ° % organic solvent concentration in mixtures. At higher concentrations certain deviations, especially for acetone and dioxane mixtures, were observed (Fig. 4)- Changes in positions of fluorescent spectral maxima, as too insignificant, were not used for comparisions. For q)- and ET-values, and for shifting m a x i m u m absorption coefficients, when plotting against dielectric constant values of mixtures containing more than 5 ° % of organic solvent, Biochim. Biophys. Acta, lO2 (1965) 289-300

290

J. KOZIOL, E. KNOBLOCH

a lack of linearity was observed, greater for acetone and dioxane t h a n for ethanolcontaining mixtures. I n the course of further investigation, it was found t h a t # , ET and absorbancy values are m u c h better correlated with the so-called Z-values derived b y KOSOWER11 as an empirical measure of solvent polarity.

7o!

Dloo °

65 eAloo

6O

65 Dgo•

5O

¢. lo 2

Ago

45 A80

4O ,D95 • A95

35

AoT/ .o

DgO

3O DSo.~Ego D

25

~Eso--

]EIo HOH

,o.o¢~:o

E ~ a 5

2O I

75

¢7

;9

[

81

Et

J7

I

79

I

81

8'3

I

8s

Fig. 4- Correlation b e t w e e n ET- a n d (/)-values for riboflavin (left curves) a n d l u m i f l a v i n (right curves) s o l u t i o n s c o n t a i n i n g e t h a n o l (E), a c e t o n e (A) a n d d i o x a n e (D) in m i x t u r e s w i t h water. N u m b e r s i n d i c a t e p e r c e n t a g e (v/v) o r g a n i c s o l v e n t c o n c e n t r a t i o n s .

The correlation obtained for ~b-values of lumiflavin solutions in acetone and dioxane mixtures were found to be linear with Z-values. (Z-values for higher dioxane concentrations than 9 ° % were not available.) For q~-values of riboflavin solutions in all solvent mixtures, and for lumiflavin in ethanol mixtures, a lack of linearity was observed (Fig. 5). W h e n ET-values for shifting absorption m a x i m u m in near ultraviolet were plotted against Z-values, straight lines were obtained for riboflavin and lumiflavin solutions in all mixtures of organic solvent with water. Only for the highest concentrations of acetone in lumiflavin solutions was a deviation seen (Fig. 6). Parallel to the changes of position of absorption m a x i m u m considerable alterations in absorbancy were also observed. The observed values for absorbancy were used as a relative empirical measure of the association degree of the two flavins with solvents in all mixtures. These absorbancy values plotted against Z-values showed Biochim. Biophys. Acta, lO2 (1965) 289-300

SPECTROSCOPY OF RIBOFLAVIN AND LUMIFLAVIN

297

only rough linearity for all solutions, with considerable deviation for lumiflavin in pure acetone (Fig. 7). When absorbancy values are plotted against qS-values, linearity can be seen only in mixtures containing not more than 80 % of organic solvent.

7C

65

•(Dl\ oo?) \

\

\

\

\ \ "X~oo \\\

60

55

5O

o.o

¢.10 2 45

E,oo~\

.,o~\~,O,o

4()

35

\,ooo

3°t

b

",o\\ t.~Bo \ ~ -~e go~ E8 0 E7 0

25

I

60

65

I

70

~

75

I

Z

80

I

85

--

a

I

90

Fig. 5. Correlation b e t w e e n gi- a n d Z - v a l u e s for r i b o f l a v i n (a) a n d l u m i f l a v i n (b) s o l u t i o n s cont a i n i n g e t h a n o l (E), a c e t o n e (A) a n d d i o x a n e (D) in m i x t u r e s w i t h water. N u m b e r s i n d i c a t e percentage (v/v) o r g a n i c s o l v e n t c o n c e n t r a t i o n s . ~ - v a l u e s for 6 0 % a n d 70 % m i x t u r e s are e s t i m a t e d .

The lack of full correlation, described above, between qs_ and ET-values, and also between q5 and Z, q~ and A, A and Z, and to some extent also ET and Z for the riboflavin solutions with highest organic solvent concentrations in mixtures, and especially for lumiflavin solutions in pure solvents, cannot be simply explained. It can, however, be considered as the result of other changes in the state of flavin molecules, which are not registered by measuring shifts of absorption maximum or absorbancies, although they can be partly detected by means of qS-values. It can be seen in lumiflavin solutions in acetone and dioxane mixtures when correlated with Z-values (Fig. 5). When other parameters were correlated among each other and with Z-values, lumiflavin in these solutions always showed the greatest deviations from linearity. According to results obtained by KOSOWERlz and general considerations given by PULLMAN AND PULLMAN13 on spectral absorption by flavin molecules in the near ultraviolet, its relatively high molar extinction coefficient seems to be connected with ---+x* transition, although the m a x i m u m is in a position claimed to be characteristic for n - - + x * transitions in quinone compounds. The shift of this m a x i m u m Biochim. Biophys. Acta, lO2 (1965) 2 8 9 - 3 o o

298

j . KOZIOL, E. KNOBLOCH

towards short wavelengths parallel with a considerable decrease in degree of association of flavin molecules with solvents, results in the increase of absorbed energy and quantum yields of emitted fluorescence. 95

HOH

HOH

9O

85

E,o\\

Z 80

75 70

65

~

'

47'

49'

8'1 ' 8'3'

Et

7'7' ~'~1

' L~'

45

Fig. 6. Correlation b e t w e e n ET- a n d Z - v a l u e s for riboflavin (left curves) a n d l u m i f l a v i n (right curves) s o l u t i o n s c o n t a i n i n g e t h a n o l (E), a c e t o n e (A) a n d d i o x a n e (D) in m i x t u r e s w i t h water. N u m b e r s i n d i c a t e p e r c e n t a g e (v/v) o r g a n i c s o l v e n t c o n c e n t r a t i o n s . E T - v a l u e s for 7 ° % m i x t u r e s are e s t i m a t e d . HOH

95

HOH

90 85

/.Eso

E,o./

D~O..,~AJ o

Z

Ego..,"~o

~

80

DgO./~,Ago

/2~o

/

,DgO

75 7C 65

"AIo0

a

2do

2;o

A

b

3bo

3;o

Fig. 7- Correlation between absorbaney values (.4) and Z-values for riboflavin (a) and lumiflavin (b) solutions containing ethanol (E), acetone (A) and dioxane (D) in mixtures with water (v/v). N u m b e r s indicate organic solvent concentrations. Absorbancy values for 7 ° ~o mixtures are estimated.

It can be considered that increasing frequency of light absorbed at the m a x i m u m in the near ultraviolet, which corresponds to the transitions to the second singlet 14 (or rather triplet)is, in excited state, leads to an increase in percentage of this structure, which must result in increasing the probability and rate of photodecomposition. In fact, in all solvent mixtures investigated, riboflavin photolability increased several Biochim. Biophys. Acta, lO2 (1965) 289-300

SPECTROSCOPY

OF R I B O F L A V I N AND L U M I F L A V I N

299

times when compared with the photolability in aqueous solution. Photolysed riboflavin solutions were examined chromatographically, and the following decomposition products were found: in water, lumiflavin and lumichrome; in ethanol, acetone and dioxane, lumichrome. However, lumiflavin, which is generally considered to be photostabile, having ribityl chain wich hydroxyl groups, was also rapidly photodecomposed in all organic solvents examined. In photolysed lumiflavin solutions, lumichrome and residual quantities of lumiflavin were found chromatographically. Riboflavin and lumiflavin, normally considered as electron-deficient compounds TM, need sources of hydrogen atoms (and/or electrons) 14 for photoreduction. Their role can probably be played for riboflavin autocatalytically b y hydrogen atoms from its ribityl side chain14,15. The rates of photodecomposition of riboflavin in all three organic solvent mixtures with water are, therefore, almost the same. In the case of lnmiflavin the rate of decomposition evidently depends on the degree of association with solvent molecules, but it is not clear from which kind of source the necessary hydrogen atoms (or electrons) are derived. I t has been shown that light-excited molecules can rapidly acquire a proton ~. The difference observedin this work between riboflavin and lumiflavin sensitivity to the nature of the solvent is most probably due to the effect of riboflavin's ribityl side chain and its probable inductive influence on the isoalloxazine part of the molecule. As the near-ultraviolet m a x i m u m shifts, and changes in absorbancies obtained for riboflavin and for lumiflavin are comparable, changes in quantum yields of fluorescence are significantly greater for lumiflavin in corresponding solvent mixtures. In experiments on photodecomposition of flavins in organic solvents TM it was found that the values reported here for positions of the near-ultraviolet m a x i m u m and quantum yields of fluorescence are near the limits. Riboflavin and lumiflavin solutions in ethanol or dioxane mixtures, with cyclohexane in a concentration limited b y both flavins' solubility, showed such a small increase in absorption m a x i m a shifts and quantum yields of fluorescence from such an increase of photolability that precise measurements of absorption and fluorescence spectra proved impossible. Thus, it m a y be assumed that absorption and fluorescence emission spectra obtained for riboflavin in 95 % and 97-5 % dioxane mixtures with water, and for lumiflavin in pure dioxane, represent virtually the true spectra of both flavin molecules, almost undissociated or weakly associated with solvent molecules. I t seems probable that the true spectra of flavins are unobtainable. From the observations discussed above a very important role of the riboflavin molecule association with water molecules in biological system can be emphasized. As the changes observed in absorption spectra of riboflavin and lumiflavin parallel with changes in water content in solvent mixtures are almost of the same degree, it seems to us that the solvent molecules are associated principally to the isoalloxazine ring. The mode in which solvent molecules associate with flavins is not clear. I t seems t h a t hydrogen bonds m a y exist, but formation of the charge-transfer complexes cannot be excluded. REFERENCES I A. KOZIOLOWA, Prace z Zakresu Towaroznawstwa i Chemii, Zeszyty Naukowe W S A Pozna¢~, Ser. I., 14 (1964) 39. 2 J. KOZlOL, Prace z Zakresu Towaroznawstwa i Chemii, Zeszyty Naukowe W S A Pozna~, Ser. I., in the press.

Biochim. Biophys. Acta, lO2 (1965) 2 8 9 - 3 o o

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J. KOZIOL, E. KNOBLOCH

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Biochim. Biophys. Acta, lO2 (1965) 289-3oo