Fluorescence and the structure of proteins VI. Non-fluorescent phenol-amide complexes

536

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 45228 FLUORESCENCE AND T H E STRUCTURE OF P R O T E I N S VI. NON-FLUORESCENT P H E N O L - A M I D E COMPLEXES ROBERT

W. COWGILL

Department of Biochemistry, Bowman Gray School of Medicine, Winston-Salem, N.C. (U.S.A.)

(Received March 2nd, 1965)

SUMMARY

Tyrosine derivatives as well as other phenols form non-fluorescent complexes with amides in non-polar solvents. For example, the association constant for the I : I complex of phenol and N , N - d i m e t h y l a c e t a m i d e in n-hexane is 285 1/mole at 25 °, A F ° = --3.34 kcal/mole and A H = --4.45 kcal/mole. Formation of the complex leads to loss of fluorescence and a characteristic red shift in the absorption spectrum of phenol; other phenolic compounds with unsubstituted and sterically unobstructed hydroxyl groups show analogous behavior. The loss of fluorescence arises from internal energy degradation within the complex. In more polar solvents, association is weaker and at higher amide concentrations (O.l-O.2 M) in the latter solvents intermolecular collisional quenching also could be observed.

INTRODUCTION

The average fluorescence efficiency of tyrosyl residues is low in native bovine pancreatic ribonuclease. Upon denaturation, the fluorescence increases to a level comparable to that for isolated tyrosyl residues in a polypeptide chain1, 2. A tentative explanation for this phenomenon is that fluorescence of some of the tyrosyl residues is diminished because of the environment and/or chemical bonding of the phenolic rings within the native protein. The present observations were made during a search for circumstances which diminish fluorescence of phenolic compounds and which might serve as a model for conditions within the native protein. The significance of these studies for protein structure has been discussed in a preliminary communication ~. Among the reactions considered in the above-mentioned survey was the hydrogen-bonded association of phenols with amides in non-polar solvents. Both the ultraviolet and infrared absorption spectra change upon association and the reaction has been studied spectrophotometrically by a number of investigators 4 6. Loss of fluorescence of the phenol upon association, as described in this paper, is even more dramatic and constitutes a convenient measure of complex formation, quite aside from its possible bearing on non-fluorescent tyrosyl residues in proteins. Biochim. Biophys. Acta, lO9

(1965)

536-543

537

NON-FLUORESCENT PHENOL--AMIDE COMPLEXES EXPERIMENTAL PROCEDURES

Chemicals The amides were obtained from Eastman Chem. Co. and were recrystallized from benzene or distilled in vacuum before use. All organic solvents were supplied by Matheson, Coleman and Bell Co. as spectro-grade quality and were found free of fluorescing or light-absorbing impurities in the wavelength region of interest.

Methods Fluorescence and absorbance were measured by conventional methods l&

Mathematical derivations (I) Determination of the number of molecules (n) of phenol bound per amide molecule in the complex: n phenol + amide ~ (phenol)n-amide g

[c]

(~)

[Phi n [Am] or log [c] = n log [ P h i + log K [Am~

(2)

where [cI = concentration of complex at equilibrium, [Phi = concentration of free phenol and [Am~ = concentration of free amide. (2) Calculation of the association constant, K, from fluorescence data: For the case n = I, Eqn. I m a y be written, K=

[c] ( [ P h ] t - - [c]) ([Am~t - - [c])

(3)

where ~Ph]t and [Am]t represent the total concentrations of phenol and amide respectively in the solution. Under the experimental conditions of the present study, [Am] t is a thousand-fold greater than [c] and Eqn. 3 may be simplified to: [c] K =

(4)

( [ P h ] t - - [c]) [Am~t

RE represent fluorescence of unassociated phenol at intermediate stages of association (the associated phenol is observed to be non-fluorescent; see Reaction d in DISCUSSION). Then [ P h l t - - [ c I = [PhltRE/REt and [cI = [Ph~t ( I - - R E / R E d. Substitution of these values in Eqn. 4 yields: L e t R E t represent the relative emission when the total phenol is free and

K =

[ P h ] t (I - - R E / R E t ) [Ph]t (RE/REt) [AmJt

(5)

or RETIRE :

I + K [Am~t

(6)

(3) Calculation of K from absorbance data: The simplifying assumption for passage from Eqn. 3 to 4 is not valid for conditions employed in the spectrophotometric study and Eqn. 3 was modified as follows. A maximum in the difference spectrum between free and associated phenol occurs at 282 m# in n-hexane and change in absorbance (AA ~s2) is a measure of association. Biochim. Biophys. Acta, lO9 (1965) 536-543

538

R.W. COWGILL AA2s2 -- Ec[c] - - Eph[cl -- AE2s2[c]

(7)

where AE2s 2 is the difference in molar e x t i n c t i o n coefficients between free a n d associated phenol at 282 m # (the small absorbance b y amide at 282 mff is compensated b y m a i n t a i n i n g the same c o n c e n t r a t i o n of amide in b o t h reference a n d sample b e a m s of the spectrophotometer). T h e n [c] = A A 2s2/AE2s2 a n d K =

AA2s2/AE2a2 ([Ph]t - - AA282/AE282) ([Am]t A-- A282/AEs82)

(8)

U p o n r e a r r a n g e m e n t of terms in Eqn. 8, [Ph]t

I

(

I

[Ph]t

AA2s2 ~

__~__

RESULTS Determination of n, the number of phenolic molecules associated per amide molecule I n the e x p e r i m e n t s u m m a r i z e d in Fig. I, variable a m o u n t s (i0 5-10 -4 M) of phenol were m i x e d with a fixed a m o u n t (0.004 M) of N , N - d i m e t h y l a c e t a m i d e in n-hexane. The a m o u n t s of free a n d associated phenol could be d e t e r m i n e d from the relative fluorescence of these solutions in the presence a n d absence of dimethylacetamide. The solid line in Fig. I corresponds to Eqn. 2 when n = I ; the broken line if n = 2 (log K [Am] is assumed c o n s t a n t in this plot since the c o n c e n t r a t i o n of amide [Am~t is one hundred-fold greater t h a n the c o n c e n t r a t i o n of complex). I I

I

I

,ogEcl[

.///n= 2/,./ n75/o

-5.5

I

I - -

i

/

n- Hexane

3 RE t

-

!

A

Butyle~her

11~/~'/

- 5.0

o,] /'

--4.5 1 ~

/ I 4.5

~

n-Butanol

! __1 - 5.0

'og [Ph]

I -5.5

~__

[ ...... 002

I 0.06

Molar c o n c e n t r a t i o n

i___ 0.10 of d i m e t h y [ a c e t o m i d e

Fig. I. Determination of the number (n) of phenol molecules associated per dimethylacetamide molecule. Lines were drawn to fit Eqn. 2 for slope n = i and n = 2. Fig. 2. Ratio of fluorescence of phenol in the absence (RE:) and in the presence RE of the amide at 25° in the solvents indicated. Biochim. Biophys. Acta, lO9 (1965) 536-543

539

NON-FLUORESCENT PHENOL--AMIDE COMPLEXES TABLE I ASSOCIATION

CONSTANTS

Solvent

FOR

PHENOL

PLUS

N,N-DIMETHYLACETAMIDE

AT

25 °

K

n-Hexane C a r b o n tetrachloride n - B u t y l ether Chloroform p-Dioxane n-Butanol

Fluorescence data

A bsorbance data

285 --* 17"* --* 8** 0. 5 * *

275 18o*** (I5)§ (I5) § (IO)§

--

* This solvent q u e n c h e s fluorescence. ** This value m a y be high because of some collisional q u e n c h i n g of fluorescence; see DISCUSSION. *** MIZUSHIMA et al. 4 h a v e r e p o r t e d values of 270 at 2o ° a n d 136 at 3 °0 from infrared data. § Association was too w e a k for accurate d e t e r m i n a t i o n from a b s o r b a n c e data.

TABLE II MOLAR THE

CONCENTRATIONS

PHENOLIC

COMPOUND

OF AMIDES

FOR

LOSS

OF HALF

THE

FLUORESCENCE

OF

AT 25 °

A mide

Fluorescent compound

Acetamide Acetamide N-Methylacetamide N-Methylacetamide N, N - D i m e t h y l a c e t a m i d e N, N - D i m e t h y l a c e t a m i d e N,N-Dimethylacetamide N,N-Dimethylacetamide N, N - D i m e t h y l p r o p i o n a m i d e e-Caprolactam

Solvent

Phenol N-Acetyltyrosine ethyl ester Phenol N-Acetyltyrosine ethyl ester Phenol N-Acetyltyrosine ethyl ester

3,5-Di(tert.-butyl)phenol 2,5-Di(tert.-butyl)phenot Phenol Phenol

n-Hexane

n-Butyl ether

p-Dioxane

n-Butanol

--

o.Io

o.14

o.82

---

o. 15 o.o8

o.2o o. I6

1.6 --

-o.oo35

o. 17 0.06

o.25 o.12

-2.0

-0.0085 O.Ol5 0.005 o.o25

o. 16 --0.09 --

o. 19 --o.15 o.Io

------

D e t e r m i n a t i o n o f the a s s o c i a t i o n c o n s t a n t ( K ) The association constant

could be calculated from Eqn. 6 which

is b a s e d

on

t h e l o s s o f f l u o r e s c e n c e of t h e p h e n o l u p o n a s s o c i a t i o n . T h i s r e l a t i o n s h i p is s h o w n i n F i g . 2 a n d s l o p e s of t h e l i n e s y i e l d v a l u e s o f K w h i c h a r e c o m p i l e d i n T a b l e I . T h e concentration olic c o m p o u n d s

(c0.5) of o t h e r a m i d e s r e q u i r e d t o h a l f q u e n c h t h e f l u o r e s c e n c e of p h e n are listed in Table II. The reason for re porting the s e re s ults in te rm s

o f qo.5 r a t h e r t h a n K w i l l b e d i s c u s s e d b e l o w . M a r k e d s h i f t s i n t h e u l t r a v i o l e t s p e c t r a of p h e n o l i c c o m p o u n d s

also occur upon

Biochim. Biophys. Acta, lO9 (1965) 536-543

R. W. COWGILL

540

l E

iI

/'

'

800 ]

2000

J

t

I

I

600

-

I I

//,,1 /)/

I000

[Am]t

'~/ '', V ',

i /

4OO

I I I I I t I

\ 0

I 260

I

\

\

I

I

0

280 Wavelength

/

2OO

0

/

1

(m~)

I

3

2

[Ph]t/z~A2e 2 x 103

Fig. 3- A b s o r p t i o n s p e c t r u m of p h e n o l in n - h e x a n e in t h e a b s e n c e (. . . . ) a n d in t h e presence ( ) of excess (0.2 M) d i m e t h y l a c e t a m i d e ; E d e s i g n a t e s t h e m o l a r e x t i n c t i o n coefficient. Fig. 4. D e t e r m i n a t i o n of K f r o m a b s o r b a n c e c h a n g e s for p h e n o l plus variable a m o u n t s of dim e t h y l a c e t a m i d e in n - h e x a n e . AE2s 2 = 159o; [Phil = 3.2' lO -4 M; 25 °. See E q n . 9.

I

I

,o0 K = - , . @ % ( ~ )

I

0.15

+ C

&H=-4.45 kcol/mole

/ ~

0,10

2.7

qo.5

log K

0.05

2.5

0.00E

2.3

0.003 3.2

3.4 ~/T x 103

3.6

f 20

60

100

Tern perature

Fig. 5. Effect of t e m p e r a t u r e on K for p h e n o l plus d i m e t h y l a c e t a m i d e in n - h e x a n e . Fig. 6. Effect of t e m p e r a t u r e on q0.5 for p h e n o l plus d i m e t h y l a c e t a m i d e in n - h e x a n e ( O - - O ) , n-butyl ether (0--0) a n d p - d i o x a n e ( X - - × ) ; a n d for p h e n o l plus p r o p i o n a m i d e in p - d i o x a n e

(A--A). Biochim. Biophys. Acta, lO9 (1965) 536-543

N O N - F L U O R E S C E N T PHENOL--AMIDE COMPLEXES

541

association with amides, as illustrated in Fig. 3, and K may be calculated from spectrophotometric data by means of Eqn. 9. The slope of the line in Fig. 4 equals I

K. AE2s~

[Ph]t AE~s~

AA282 (AE2s2)2

from Eqn. 9. Under the conditions for the experiment in Fig. 4, the last term, AA~s2/(AE~s2) 2 is less than one per cent of the total value and has been neglected in these calculations; all other terms are constant and all are known except K. For example, see Fig. 4 and the compilation in Table I.

Effect of temperature upon K and the fluorescence quenching phenomenon A graph of log K vs. I / T for phenol plus dimethylacetamide in n-hexane is linear with a positive slope (Fig. 5). In more polar solvents, anomalous curves were observed and some of these are represented in Fig. 6. In the latter figure, q0.5 is plotted vs. temperature for reasons discussed below. DISCUSSION The fluorescence of phenols has been described by a number of investigatorsL Upon addition of an amide to the phenol in an organic solvent, the fluorescence was lost. This loss of fluorescence did not occur if the dissociable hydrogen atom was replaced by a methyl group, as in anisole; nor did it occur with phenol when the carbonyl group of the amide was blocked. That is, e-caprolactam effectively abolished the fluorescence of phenol but O-methylcaprolactim did not. Infrared spectral studies 4-~ also indicate that the association of phenols with amides involes the formation of a hydrogen bond between the hydroxyl and carbonyl groups; and the reaction appears to be as follows:

<--~-O-H + 0 = C- ~/ NRR 2 -~ ~- L > 0 - H / •.0=C~R/NR~ -

-

[,]

where R may be H or an aliphatic group. The generality of the reaction is illustrated by the compilation of ADELMAN8. Values of K = 275-285 1/mole for association of phenol with N,N-dimethylacetamide in n-hexane at 25 °, as calculated from fluorescence and absorbance data, are comparable with values reported for the association of these compounds in isooctane and cyclohexane by MIZUSHIMAet alA. In more polar solvents, association was weaker (Table I) and could not be detected in aqueous solutions. The principal effect of these polar solvents appears to be competition by the solvent as a hydrogenbond donor or acceptor. For example, TAKAHASHIAND Lt 9, have shown that alcohols serve as donors for hydrogen-bonded association with amides; also, ketones and ethers 4 as well as dioxanO ° are hydrogen-bond acceptors for association with phenols. Closely related to this competitive hydrogen-bonding phenomenon is the dimerization of amides; and it has been observed that dimerization increases in more non-polar solvents u-13. Because of dimerization and the attendant uncertainty regarding the effective concentrations of the amides for association with phenols, K values were not calculated for unsubstituted or monosubstituted amides. Instead,

Biochim. Biophys. Acta, lO9 (1965) 536-543

542

R. W. COWGILL

t h e c o n c e n t r a t i o n of a m i d e for loss of half the fluorescence was d e t e r m i n e d from g r a p h s similar to Fig. 2 a n d t h e values are a s s e m b l e d in T a b l e I I . (For the N , N d i s u b s t i t u t e d amides, K = I/q0.5.) T h e loss of fluorescence of phenols in the presence of a m i d e s could be either b y collisional quenching of the e x c i t e d s t a t e as r e p r e s e n t e d in R e a c t i o n c or b y form a t i o n of a non-fluorescent c o m p l e x as r e p r e s e n t e d in R e a c t i o n s d a n d e. (An e x a m p l e of the former process is the collisional quenching of phenols in aqueous solution b y c a r b o x y l i c acids. 14) The fluorescence d a t a when p l o t t e d as in Fig. 2 could c o r r e s p o n d Ph + hv ~

Ph* (formation of the excited state)

(a)

Ph* --~ Ph + hv' (emission of fluorescence)

(b)

Ph* + Am --+ energy degradation by transient collision

(c)

Ph + A m ~ P h A m

(d)

PhAm + hv --+ energy degradation by internal conversion processes in the static complex

(e)

to either t h e S t e r n - V o l m e r f o r m u l a t i o n for collisional quenching R E T I R E = i + k [Am~, where k is a composite of t h e r a t e c o n s t a n t s of R e a c t i o n s a - c or to f o r m a t i o n of a non-fluorescent c o m p l e x as r e p r e s e n t e d b y Eqn. 6 a n d R e a c t i o n s d a n d e. F o r phenol p l u s d i m e t h y l a c e t a m i d e in hexane, the l a t t e r process was t h e correct one. This was e s t a b l i s h e d b y the spectral evidence t h a t a long-lived association c o m p l e x does exist a n d b y a g r e e m e n t between a b s o r b a n c e a n d fluorescence d a t a for t h e value of the association constant. I n circumstances less f a v o r a b l e for R e a c t i o n d, for e x a m p l e the association of phenol a n d d i m e t h y l a c e t a m i d e in a more p o l a r solvent or the r e p l a c e m e n t of d i m e t h y l a c e t a m i d e b y an a m i d e which can dimerize, the concent r a t i o n of a m i d e necessary for loss of fluorescence is c o n s i d e r a b l y greater. U n d e r these circumstances, one m i g h t e x p e c t to find collisional quenching between nona s s o c i a t e d phenol a n d a m i d e molecules in a d d i t i o n to t h e i n t r a m o l e c u l a r quenching t h a t occurs in t h e r e l a t i v e l y s t a b l e complex. The effect of t e m p e r a t u r e u p o n t h e values of q0.5 indicates t h a t b o t h these processes can occur. On one hand, increase of t e m p e r a t u r e would decrease t h e degree of association a n d necessitate g r e a t e r c o n c e n t r a t i o n s of a m i d e for quenching; this is i l l u s t r a t e d in Fig. 6, for n - h e x a n e as solvent. On t h e o t h e r hand, increase of t e m p e r a t u r e would increase the collision f r e q u e n c y b e t w e e n n o n - a s s o c i a t e d molecules a n d t h u s decrease the c o n c e n t r a t i o n of a m i d e necessary for quenching b y this process. A t r e n d in this l a t t e r direction also is o b s e r v e d in Fig. 6 for more p o l a r solvents in which r e l a t i v e l y high concent r a t i o n s of a m i d e are present. I n the case of phenol p l u s p r o p i o n a m i d e in dioxane t h e slope of the curve in Fig. 6 is reversed a n d a collisional quenching process pred o m i n a t e s . T h e high c o n c e n t r a t i o n of anaide (approx. o . I M) r e q u i r e d for collisional quenching indicates t h a t t h e process is inefficient. This m i g h t be a t t r i b u t e d either to a h e a t of a c t i v a t i o n for quenching or to a strict collisional o r i e n t a t i o n r e q u i r e m e n t ; if t h e l a t t e r is so, t r a n s i e n t h y d r o g e n - b o n d i n g m a y also be a r e q u i r e m e n t for t h e collisional quenching process. The fact t h a t fluorescence of anisole is not quenched b y collision w i t h amides is evidence t h a t this l a t t e r p r o p o s a l m a y be true. Biochim. Biophys. Xcta, lO9 (1965) 536-543

NON-FLUORESCENT PHENOL--AMIDE COMPLEXES

543

ACKNOWLEDGEMENTS I d e e p l y a p p r e c i a t e t h e c a r e f u l t e c h n i c a l a s s i s t a n c e of Mr. JOHN B. TINKLER. This investigation was supported

by a U.S. Public Health

Service research grant

( G M - I o 5 1 5 - o 2 ) f r o m t h e N a t i o n a l I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s .

REFERENCES

i 2 3 4 5 6 7 8 9 io II 12 13 14

R. W. COWGILL,Arch. Biochem. Biophys., ioo (1963) 36. R. W. COWGILL, Arch. Biochem. Biophys., lO 4 (1964) 84. R. W. COWGILL, Biochem. Biophys. Res. Commun., 16 (1964) 332. S. MIZUSHIMA, M. TSUBOI, T. SHIMANOUCHI AND Y. TSUDA, Spectrochim. Acta, 7 (1955) IOO. T. GRAMSTAD AND W. J. FUGLEVlK, Acta Chem. Scand., 16 (1962) 1369. M. D. JOESTEN AND R. S. DRAGO, J. Am. Chem. Soc., 84 (I962) 2037, 2696 and 3817. S. UDENFRIEND, Fluorescence Analysis in Biology and Medicine, Academic Press, New York, 1962, p. 32. R. L. ADELMAN, J. Org. Chem., 29 (1964) 1837. F. TAKAHASHI AND N. C. LI, J. Phys. Chem., 41 (1964) 2136. S. NAGAKURAAND H. BABA, J. Am. Chem. Soc., 74 (1952) 5693 • S. MIZUSHIMA, Advan. Protein Chem., 9 (1954) 299. I. M. KLOTZ AND J. S. FRANZEN, J. Am. Chem. Soc., 84 (1962) 3461. J. S. FRANZEN AND R. E. STEPHENS, Biochemistry, 2 (1963) 1321. F. WOLD AND G. WEBER, Federation Proc., 22 (1963) 348.

Biochim. Biophys. Acta, lO9 (I965) 536-543