249
Sugar Effects on Amino Acid and Model Peptides
while no T L was observed from fructose, mannose, galactose, fucose, or cellobiose. Thus, the presence or absence of T L is probably a function of the crystal structure or the packing of molecules in a crystal and not solely a function of the size of a crystal. We have observed similar behavior of the T L of N-acetylanthranilic acid and its derivatives. Minor changes of the substituents which barely affect the luminescence properties of the molecule prohibit the TL.1° Work is in progress to determine the structural factors necessary for a crystal to be triboluminescent.
Acknowledgment. The Army Research Office, Durham, is gratefully acknowledged for support of this work.
References and Notes (1) (a) Camille and Henry Dreyfus Teacher-Scholar, 1974-1979. (b) Contribution No. 3494. (2) (a) F. Bacon, “Of the Advancement of Learning”, 1605, G. W. Kitchin, Ed., J. M. Dent and Sons, London, 1915. (b) E. N. Harvey, “A History of Luminescence”, American Philosophical Society, Philadelphia, Pa., 1957, Chapter 10. (3) J. I. Zink and W. Klimt, J. Am. Chem. Soc.,96, 4690 (1974). (4) J. I. Zink and W. C. Kaska, J. Am. Chem. SOC.,95, 7510 (1973). (5) J. I. Zink, J. Am. Chem. SOC.,96, 6775 (1974). (6) J. I. Zink, Inorg. Chem., 14, 555 (1975). (7) C. R. Hurt, N. Mcavoy, S. Bjorklund, and N. Fillipescu, Nature (London), 212, 179 (1966). (8) J. I. Zink, Chem. Phys., Lett., 32, 236 (1975). (9) G. Herzberg, “Molecular Spectra and Molecular Structure. I. Spectra of Diatomic Molecules”, 2nd ed., Van Nostrand, New York, N.Y., 1950, p 32. (IO) W. C. Kaska, G. Hardy, and J. I. Zink, unpublished observatians.
Effects of Sugar Solutions on the Activity Coefficients of Aromatic Amino Acids and Their N-Acetyl Ethyl Esters T. S. Lakshml and P. K. NandP Protein Technology Discipline, Central Food Technological Research Institute, Mysore 5700 13,India (Received May 14, 1975)
Activity coefficients of N-acetyl ethyl esters of phenylalanine, tyrosine, and tryptophan have been determined in aqueous sucrose and glucose solutions. Similar measurements have been carried out with phenylalanine, tyrosine, and tryptophan in sucrose solution. The compounds show increased activity coefficients (decrease in solubility or “sugaring o u t ” ) in the solutions studied. The molar free energy of transfer, U t r , of these compounds from water to sugar solutions has positive values and indicates increased hydrophobicity in sugar solutions. The AFtr value is found to remain unaltered over a considerable range of temperature. These observations have been utilized to explain the thermal stability of protein in aqueous sugar solutions. Prevention of heat coagulation of ovalbumin in sucrose solution was recorded a long time back.l Similar observations of the stability of ovalbumin and serum proteins in sugar solutions have been r e p ~ r t e d . ~Simpson -~ and Kauzmann observed that the extent of ovalbumin denaturation in urea was reduced in presence of s u ~ r o s eGerlsma .~ and Stuur observed that the melting temperature of both ribonuclease and lysozyme increases in sorbitol solution indicating their increased thermal stabilities in the solution.6 Recently we have observed that the heat coagulation of aglobulin, the major protein component of sesame seed ( S e s a m u m indicum L.) proteins, is prevented in sucrose and glucose solutions (unpublished work). The manner in which sugar induces the increased heat stability of proteins or reduces the extent of denaturation by other reagents is not known. Studies during past few years have indicated how the groups which are mainly involved in the denaturation process of a protein molecule would behave in different solvent systems, e.g., simple electrolytes, urea, guanidine hydrochloride, and tetralkylammonium salt solution^.^-^^ This information was obtained from a study of the activity coefficients of model compounds which are representative of different groups in a protein molecule. In our previous studies we chose blocked
amino acid ethyl esters, e.g., CH3CQNHCW(R)CQQC2H5, R being a nonpolar side chain. Here we report measurements of the activity coefficient of the above compounds where R is phenylalanyl, tyrosyl, and tryptophanyl side groups in sucrose and glucose and also aromatic amino acids in sucrose solutions. The phenylalanyl derivative has also been studied in aqueous glycerol, ethylene glycol, and 1-butanol solutions. Temperature effect on the activity coefficient of the compounds has also been determined to obtain a better insight into the mechanism of sugar effects on model compounds and hence protein.
Experimental Section Materials and Methods. N -Acetyl-L-phenylalanine ethyl ester (Sigma Chemical Co.), N-acetyl-L-tyrosine ethyl ester (Grade I, Cyclo Chemical Corp.), N-acetyl-L-tryptophan ethyl ester (Grade I, Cyclo Chemical Corp.), L-phenylalanine (BDH), L-tyrosine (E. Merck), and L-tryptophan (E. Merck) were used. Solubility phase curves for these compounds showed the absence of impurities. Sucrose and glucose were reagent grade chemicals. Fractions of ethylene glycol (E. Merck) and I-butanol (E. Merck) distilling respectively at 115-117 and 194-196O were used; BDH glycerol was used. Glass distilled water was used. The Journal of Physical Chemlstfy, Voi. 80,No. 3, 7976
T. S. Lakshml and,P. K. Nand1
250
The solutes were equilibrated with the solvents in 10-ml capacity tubes sealed with Teflon-lined screw caps. For the measurements at 28 f 0.1 and 5 f 1’ the tubes were submerged in a water bath in a rotating rack, and mixing was accomplished by rotating tubes end-over-end a t 35-40 rpm. The solubility measurements a t 40 (f0.2’) and 55’ (f0.3’) were carried out in an incubator shaker (New Brunswick Scientific Co., N.J.) at 180 oscillation/min. In general the period of equilibration was 5 days at 5’, 60 h a t 28O, and 48 h a t both 40 and 55’. The measurement at 65’ with APE was carried out by shaking the solutions intermittently for 8 h. Equilibrated solutions were filtered a t the temperatures of equilibration. Sampling of aliquots was also carried out near the equilibration temperature. The concentration of the solutes was determined by measuring the absorbance of the solution, after proper dilution, at 257 nm for Phe and APE;275 nm for Tyr and ATYE, and 278 nm for Try and ATRE in a Carl-Zeiss VSU-2 spectr~photometer.~~ Molar extinction values are APE 186 (lit. 18B9),ATYE 1335, ATRE 5445, Phe 170 (lit. 190l1), Tyr 1250 (lit. 13708), and Try 5610 (lit. 550012).The solubility values of APE in water at 5, 28, 40, 55 and 65’ are 2.55, 4.13, 6.62, 8.04, and 17.1 g/l., respectively. The solubilities of ATYE at 5 and 28’ are 1.40 and 3.48 g/l., respectively, values for ATRE a t 5 and 28’ are 0.52 and 1.47 g/l., respectively. At 28’, the solubilities of Phe, Tyr, and Try are 30.3, 0.579, and 11.0 g/l., respectively. Solubility values are reproducible within -5%. We have assumed that this range of experimental error applies also to determinations in other solvents studied here.
Results Activity coefficients of the solutes have been obtained from
fi”
= CiO/Ci~
(1)
where f i 5 is the activity coefficient of the solutes, i, in sugar or other solutions; Ci0 and Cis are the molar concentrations of i in water and the sugar solutions, respectively. Activity coefficients of the solutes have been assumed to be unity in water.13 The solubility values of the compounds in different solvents have been shown in Tables I and 11. Figure 1 shows a semilogarithmic plot of activity coefficient of APE as a function of sugar or alcohol concentration. The plots are nonlinear. In sugar solutions, the increase in the activity coefficient is observed. The increase becomes more pronounced a t higher concentrations of sugars. Glycerol and glycol also increase the activity coefficients, but to a smaller extent compared to sugars. In contrast in 1-butanol the activity coefficient decreases. Figure 2 shows similar plots of activity coefficients of the compounds studied here in sucrose solution. At a constant sucrose concentration (>1M particularly), the increase in the activity coefficient follows the order: ATYE > APE > ATRE, Try > Tyr, Phe. The same trend is observed for the esters in glucose solution. Analogous to salting out phenomenon, where the solubility decreases and correspondingly activity coefficient increases, the present observation of increased activity coefficient of the amino acids and esters can be considered as “sugaring out” phenomenon. The relative effects of different solvents on the activity coefficient of the solutes can be obtained from an equation similar to Setchnow equation in electrolyte solution, viz., log f i = K,C, where K , is the value of f i a t 1 M sugar or alThe Journal of Physical Chemistry, Vol. 80, No. 3, 1976
TABLE I : Values of the Solubility in g/l. of the N-Acetyl Aromatic Amino Acid Esters and Aromatic Amino Acids in Sucrose and Glucose Solutions at ( 2 8 ° C ) Compound Phenyl- Tyro- TryptoATYE ATRE alanine sine phan
Concn,M
APE
Water Sucrose 0.25 0.5
4.13
3.48
1.47
30.3
0.58
11.0
3.88 3.59 2.89 2.31 1.57
3.20 2.96 2.33 1.74 1.15
1.43 1.35 1.17 0.99 0.83
28.78 27.87 25.45 23.33 21.21
0.56 0.54 0.50 0.46 0.42
10.20 9.68 8.36 7.15 6.05
3.82 3.60 3.22 2.89 2.56
3.27 3.06 2.64 2.22 1.93
1.43 1.35 1.25 1.11 0.99
1.0
1.5 2.0 Glucose 0.25 0.5 1.0
1.5 2.0
4.01
0 SUCROSE
a GLUCOSE
P A
ETHYLENE GLVCOL
X ??-BUTANOL
I
I
I
2.0 M
1.0 PUGAR]
or
ELCOHOg
Figure 1. Effects of different solvents on the activity coefficients of acetyl phenylalanine ethyl ester at 28’.
coho1 concentration (C, = 1). The plots are curved and there are not many data at lower concentrations (
