Speztrochinlica Acta, 1964,Vol. 30, PP.1543to 1554.Pergatllon PressLtd. Printedin NorthernIreland
Charge transfer interactions of proteins, amino acids and a.mines in polar solvents M. A. SLIFKIN The Physical Laborat#ories, The University,
Manchester*
(Received 30 October 1963) Abstract-Spectra of solutions of proteins, amino acids and amines with chloranil in different polar solvents have been studied and recorded. It is shown that these substances cause initially the ionisation of the chloranil. Spectra of solutions of proteins, amino acids and amines with iodine in water have been studied. It is suggested that the spectral changes are consistent with the formation of protein, amino acid iodine ncomplexes. The effect of solvent polarity on the spectrum of iodine has been studied. INTRODUCTION SPECTROSCOPIC studies made of the interaction between amino acids and the electron acceptors oxygen [l], chloranil [2] and riboflavin [3] and between protein and chloranil [2], have shown that the amino acids and proteins behave like n-electron donors in a similar manner to aliphatic amines [2, 41. Some of these studies [2-41 were carried out in the moderately polar solvent 50 ‘A aqueous ethanol. Proteins and amino acids in viva are probably surrounded by very strong polarisation forces. It seems useful to examine charge transfer interactions involving proteins and amino acids in solvents of high polarity. EXPERIMENTAL
Studies have been made of the interaction of amino acids, proteins and amines with two charge acceptors, chloranil and iodine, in polar solvents. Absorption measurements were made with the SP 700 absorption spectrophotometer. Analytically pure aliphatic amines were employed. N.B.C., amino acids prepared as chromatographic standards were employed. Amino acids and proteins studied are listed in Table 1. RESULTS
AND
DISCUSSION
A solution of cu. 1O-4 M chloranil in the polar solvent dimethyl sulphoxide has a yellowish colour, unlike lop4 M chloranil in 50 % ethanol solution which is greeu. The absorption spectrum of chloranil in dimethyl sulphoxide is shown in Fig. I. The ratio of the absorption at the 285 nm peak to the shoulder at ca. 360 nm is 6: 1 whereas in the spectrum of chloranil in less polar solvents [2, 51 the ratio is of * Present address: upon Tyne 1. [l] [2] [3] [4] [5]
Department
of Physics, Rutherford
College of Technology,
M. A. SLIFKIN, Nature 193, 464 (1962). J. B. BIRKS and M. -4. SLIFKIN, Nature 197, 42 (1963). M. A. SLIFKIN, Nature 197,275 (1963). M. A. SLIFKIN, Nutwe 195,693 (1962). K. H. HAUSSER and R. S. MTJLLIKEN.J. Phys. Chem. 64, 367 (1960).
h’ewcastle
1544
11.
A.
SLIFKIN
Table 1 Amino acids and proteins glycine tryptophan hydroxyproline tyrosin alpha-alanine betaalanine fibrinogin gamma+globulin
isoleucine alanine serine leucine phenylalanine arganine trypsin bovine plasma albumin
I
.o -
8.5
.O-
.5-
.o-
,
I
300
400
I 500
Fig. 1. Absorption spectrum of chloranil in dimethyl sulphoxide.
the order of 30 : 1. Solutions of chloranil in dimethyl sulphoxide were stable for many weeks and no degradation into chloranilic acid was observed. Adding amino acid or protein to a, lo-* M solution of chloranil in dimethyl sulphoxide caused a rapid change to a deep golden colour. The spectrum of an amino acid, chloranil mixture is shown in Fig. 2. The principal features of this spectrum are the decrease of the chloranil absorption and the appearance of two new bands at 315 nm and 455 nm with minor peaks at 330 nm and 427 nm. The spectra of these amino acid, protein and chloranil mixtures were found to be quite stable. Only chromatographically pure amino acids gave these results. Analytically pure amino acids gave
Charge transfer interactions of proteins, amino acids and amines in polar solvents
1545
the same spectra as in Fig. 2 initially but over a period of several hours the spectral bands decreased and a broad structureless band at 520 nm appeared. Similar degradation of chloranil spectra have been observed and attributed to the conversion of chloranil to chloranilic acid [2].
Wavelength.
Fig. 2. Difference spectrum. Isoleucine and chloranil vs. chloranil in dimethyl sulphoxide.
The spectrum of sodium chloranil salt, i.e. the chloranil negative ion, in dimethyl sulphoxide is shown in Fig. 3. This spectrum is very similar to those of the amino acid, protein chloranil mixtures illustrated in Fig. 2. FOSTER and THOMPSON [6] have tabulated the wavelength of the peaks of the chloranil ion in various polar solvents in the wavelength region greater than 350 nm. They agree with the results given here. The negative ion was unstable in solution and over a period of several hours, the spectrum reverted to that of chloranil. Heating up chloranil solutions or mixtures of amino acid or protein and chloranil caused a complete loss of spectral structure. Similar spectra to those of Fig. 2 were obtained from mixtures of amino acids of protein and chloranil in 50 % aqueous dimethyl sulphoxide, 50 ‘A ethanolic dimethyl sulphoxide, 50% tetrahydrafuran and 75 % carbon tetrachloride in dimethyl sulphoxide. One slight difference is that in the less polar solvents the band at 427 nm is greater with respect to the 455 nm band, as in Fig. 4. No interaction was observed in distilled water, apparently due to the very low solubility of chloranil. [6] R. FOSTER
and T. J. THOMSON,
Trans.
Paraduy
Sot. 58, 860 (1962).
1.546
M.
A.
SLIFKIN
0.
0.1’
2.
z
F % E 2
0.1
8
0.05
/
0
300
400
500
600
Wavelength,
Fig.
3. Absorption
0
!
350
spectrwn of sodium dimethyl snlphoxide.
I
I
400
450
chloranil salt in
500
Wavelength,
Fig. 4. Absorption spectrum of glycine and chloranil in 50 % aqueous dimethyl sulphoxide.
Charge transfer interactions of proteins, amino acids and amines in polar solvents
1547
Freezing mixed solutions of protein and chloranil in the various solvents caused a purple precipitate to come down. The residual solution showed a marked loss of absorption. The purple precipitate was very similar to those obtained from protein chloranil mixtures in alkali 50 % ethanol [2]. The addition of aminobenzoic acids to cu. lo-* M chloranil in dimethyl sulphoxide or 50 % aqueous dimethyl sulphoxide caused a decrease in the chloranil absorption and the appearance of the negative ion spectrum. 0.31
Wavelength,
Fig. 5. Difference spectrum. Triethylamine (1 M) and chloranil (10e4 M) vs. chloranil in dimethyl sulphoxide one hour after mixing.
Studies were also made of the interaction of three aliphatic amines, ethylamine, diethylamine and triethylamine with chloranil in dimethyl sulphoxide. Immediately on adding these three amines cu. 10-l M to a lo-* M solution of chloranil in dimethyl salphoxide, the negative ion spectrum was observed. In the case of triethylamine and diethylamine the charge-transfer band [2, 71 at 650 nm and 625 nm respectively, were also observed. The spectra of these mixtures changed rapidly. With triethylamine, the spectrum changed over a period of an hour to give a broad structureless band at 530 nm and the chloranilic acid band at 520 nm, as shown in Fig. 5. With diethylamine, bands at 390 nm and 530 nm appeared as in Fig. 6. With ethylamine, new bands at 360 nm and 520 nm appeared as illustrated in Fig. 7. Similar spectra were obtained for these aliphatic amines with chloranil in 50 % dioxane. [7] S. K. CHAKRABARTY
and
A. K. CHANDRA,
Nuturwiss.
49,
206 (1962).
M. A.
1548
SLIFKIX
0.8 -
o-7-
0.6 -
x c ? % 5 .o h 0
0.5 -
0.4 -
0.3 -
0.2-
0.1 -
Wavelength, Fig.
6. Absorption
spectrum of diethylamine (1 M) and chloranil dimethyl sulphoxide one hour after mixing.
,
300
400
(lo-”
I
I
500
600
M) in
Wavelength, Fig.
7. Absorption
spectrum of ethylamine (1 M) and chloranil sulphoxide one hour after mixing.
( 10e4 M) in dimethyl
FOSTER and THOMSON [6,8, 91 have shown that various aromatic amines, in the presence of different electron acceptors, including chloranil, in polar solvents give They have suggested that n-n charge rise to the negative ion of the acceptor. transfer takes place. It has been firmly established both in this and previous studies [2> 3, 4, lo]that the aromatic amino acids and proteins behave like aliphatic electron donors and that therefore the electron transfer is n--7Tin character. [8] R. FOSTER
and T. J. THOMSON, Trans. Faraday [9] R. FOSTER and T.J. THOMSON, Trans. Faraday [IO] M. A. SLIFKIN, Nature 193,464 (1962).
SIX. Sot.
59,1059 (1963). 59,296 (1963).
Charge transfer interactions of proteins, amino acids and amines in polar solvents
1549
The ionisation of chloranil in the presence of amino acids and proteins confirm that they are electron donors. In less polar solvents [2] it was shown that the absorption band of chloranil in the n-n complex was shifted to longer wavelength. The negative ion spectrum marks the limit to which this red shift goes. These spectral shifts on complexing have been previously discussed [ 111. One interesting feature of these amine chloranil n-n complexes is that it is possible to follow the absorption band of the chloranil as it shifts to the red and changes to the negative ion spectrum on increasing the polarity of the solvent. In Figs. 2 and 3 it was shown
/ 360
380
I
1
I
I
1
I
1
400
420
440
460
480
500
520
Wavelength, Fig.
K. Absorption
spectrum of o-aminobenzoic acid and chloranil in 50 % ethanol buffered to pH 3.6.
that in the negative ion spectrum in dimethyl sulphoxide, the major band occurred at 455 nm whilst that at 427 nm was much weaker. In 50 y0 aqueous dimethyl sulphoxide, see Fig. 4, or other less polar solvents [6] the 427 nm bandincreases greatly with respect to the 455 nm band. Figure 8 shows the spectrum of o-aminobenzoic acid and chloranil in 50 o/0aqueous ethanol buffered to pH 3.6. The major band lies at 427 nm and the peak at 455 nm is very weak. There thus appears to be no abrupt change in spectrum as the electron transfer is increased with increasing polarity at the solvent. It is consequently difficult to say whether the mixtures showing the negative ion spectrum are composed of free radicals produced via charge transfer complexes as suggested by FOSTER and THOMSON[6] or are bound charge transfer complexes in which the ground state binding occurs due to the complete transfer of an electron from donor to acceptor [Y, 111. The stability of the spectra observed and the gradual transition to the negative ion spectrum suggests that the latter is the case. No other spectral features were observed in the region 270 nm to 1000 nm.’ The spectra of the protein and amino acid positive ions have never been observed but presumably lie in the far ultra-violet. [ll]
M. A. SLIFKIN, Nature
198, 1301 (1%:~).
1550
M. A.
SLIFKIN
The aliphatic amines appear initially to ionise chloranil on mixing in polar solvents but the subsequent rapid changes in spectra suggest that some chemical reaction takes place. This may be the consequence of some impurity as only analytical pure amines were used. Various other studies of aliphatic amines with charge acceptors have been made [12-141. MILLER and WYNNE-JONES [12] have suggested that the interaction of aliphatic amines with 1: 3 : 5 trinitrobenzene ca ses ionisation followed by further chemical reaction in polar solvents thus paralle 3ing the results here.
0 220
260
300
yio
380
1
420
460
I
500
I
540
Wavelength,
Fig. 9. Absorption
spectrum of iodine in water.
Solutions of iodine in de-ionized water were prepared by standing excess iodine in water for several hours until a dark brown colour developed and then decanting off. The spectrum of this solution is shown in Fig. 9. This agrees with previously published spectra [15], having peaks at 465 nm, 353 nm and 287 nm, the last two peaks being characteristic of the triiodide negative ion. KATZIN [15] has suggested that the triiodide ion, I,-, is formed from iodine in water due to impurities, in the following reactions, I 2 + Is- + I(1)
I, + I- + I,-
(2)
The 465 nm peak arises from solvated iodine. The iodine absorption peak in inert solvents is at cu. 520 nm [ 161. When excess protein or ammo acid are added to aqueous solutions of iodine, bleaching occurs. This process takes several minutes with tryptophan and protein and several hours with other amino acids. The bleaching goes more rapidly on [1Z] R. E. MILLER and VV. F. K. WYNNE-JONES, J. Chem. Sot. 2375 (1959). 1131 R. [14] R. [15] L. FIG] H.
FOSTER, J. Chem. Sot. 3508 (1959). FOSTER and R. K. MACKIE, Tetrahedron 16,119 (1961). I. KATZIN, J. Chem. F’hys. 21, 490 (1953). TSUBOMURA and R. I’. LANG, J. Am. Chem. Sot. 83, 3085 (1961).
Charge
transfer interactions of proteins, amino acids and amines in polar solvents 1551
heating. The absorption spectra of these bleached solutions were all found to be very similar, a typical example for an aliphatic amino acid being shown in Fig. 10. The proteins and aromatic amino acids displayed similar spectra but with the superposition of the protein or amino acid spectral bands. It is seen that there is a strong band with a peak at 226 nm and a very weak band with an ill-defined peak at ea. 300 nm. The iodine negative ion I- has a peak at cu. 230 nm [17]. A similar spectrum to Fig. 10 was possessed by the reaction product of ethylamine and iodine in water, ethylammonium iodide.
I.0
-
0.01
200
Fig. 10.
220
240
I
1
I
260
280
300
320
Absorption spectrum of old solution of iodine and hydroxyproline in water.
The spectra of solutions of amino acids and protein with iodine were also examined during the bleaching process. Immediately on adding amino acid or protein to aqueous iodine, there is a decrease in the band at 455 nm and an increase in the two bands at 353 nm and 287 nm. A new band appears at 226 nm. A negative peak is observed in the difference spectra at 560 nm. This is shown in Fig. 11. With time the band at 226 nm increases, the other bands all decrease until the spectrum shown in Fig. 10 results. The band at 465 nm disappears at a greater rate than that at 560 nm so that after an hour or so after mixing the band at 560 nm in the difference spectrum is lost under the 465 nm band. The changes in spectra resulting immediately on adding amino acids to aqueous iodine can be reversed by diluting with water. This is illustrated in Fig. 12. Similar results to those just described are found with mixtures of aminobenzoic acids and iodine in water. [17] A. D. AWTREY and R. E. CONNICK,
J. Am. Chem. Sm. 73, 1842 (1951).
1552
M. 0.8
A.
SLIFKIN
r-
0.7
0.6
0.5 k C_ 2 $
0.4
0 .z
0.3
0" 0.2
0.1
0
-0.1
I
200
1
240
280
Fig. 11. Difference
0.01
F I2o
280
320
I
I
I
I
360
400
440
480
520
Iodine vs. isoleucine spectrum. Immediately on mixing.
320
360
400
440
480
Wavelength,
Fig. 12. Absorption
spectra.
(a) Iodine and glycine in water. (b) Solntion (a) at, greater dilution. - - - - -
I 560
and
iodine.
Charge transfer interactions of proteins, amino acids and amines in polar solvents
1653
The effect of increasing the polarity of the solvent was also studied. The spectrum of iodine in the very polar solvent dimethyl sulphoxide is shown in Fig. 13. It is seen that the 465 nm band has completely disappeared and only the bands characteristic of I,- are seen. In mixtures of water and dimethyl sulphoxide spectra intermediate to Figs. 11 and 13 are seen.
I.6
0.6
Wavelength,
Fig. 13. Absorption
spectrum of iodine in dimethyl sulphoxide.
Similar spectra to those shown in Fig. 11 have been found by LUPINSKI and HUGGINS [ 181 to characterise the beta-carotene-iodine complex. LUPINSKI [ 191 has suggested the following scheme for the beta-carotene (C40H56) iodine interaction C,,H,,
+
21, + G,Hd+)
+ I,-
where (C,OH,,.I+) is a charge transfer complex with an excited state (C4,,H,,+.I). It has been demonstrated that the effect of amino acids and proteins on aqueous solutions of iodine can be reproduced by increasing the polarity of the solvent. The amino acids and the protein exist as polar molecules in aqueous solution and the spectral changes observed on mixing in iodine can be attributed to the increase However the aminobenzoic acids are not polar of the polarity of the solvent. molecules and would not be expected to have such an effect unless the aminobenzoic acid-iodine interaction product is polar. [181 J. H. LUPINSKI
and C. M. HUGGINS, J. Phys. Chew. 66, 2221 (1962). [19] J. H. LUPINSKI, General Electric Research Report No. 63-RL-(331C)
(1963).
M. A. SLIFKIN
1554
Lupinski’s explanation of the beta-carotene-iodine interaction would also explain the interactions observed immediately after mixing thus, amine + I+ + (amine I+) complex. The removal of I+ will cause the equilibria represented by equations (1) and (2) to go to the right and will increase the I,- concentration while decreasing the I, concentration. The reversibility of the amino-acid-iodine mixture spectrum can be explained by dilution of the amino acid lowering the polarity of the solvent. In addition, part of the reversibility may be due to the presence of an amine-iodine complex. The appearance of the 226 nm band and its continued growth in time as the other spectral bands decrease must be due to slow iodination. The iodine presumably attaches itself to the amino group as the I- ion. The removal of iodine either as I, or I- from solution will result in a decrease of the concentration of I,-, the equilibrium represented by equation (2) being moved to the left. The small peak appearing as a depression in the negative portion of the spectrum at 560 nm in Fig. 11, is possibly due to unsolvated iodine. This would suggest that an equilibrium exists in aqueous solution between solvated and unsolvated iodine, the solvated iodine greatly predominating and furthermore that the amines take up this unsolvated iodine in preference to the solvated iodine. No firm evidence has been produced that charge transfer takes place between the amino acids or proteins and iodine but in view of the results with the non-polar aminobenzoic acids and chloranil and the earlier evidence [l, 2, 31 that amino acids and proteins are good n-electron donors, it is likely that complexes of the form amino acids . I+ or proteins
1
are initially present in these solutions.
The biological implication of the ionization of electron systems has been discussed by SZENT-GYORGYI [20].
acceptors
in biological
Acknowledgements-I would like both to thank Dr. R. FOSTER (University of St. Andrews) for the generous gift of sodium chloranil salt and to acknowledge tenure of a D.S.I.R. Research Fellowship. [20] A. SZENT-GYBRGYI, Bioenergetics, Academic Press, New York (1957).