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The structure of glutaraldehyde in aqueous solution determined by ultraviolet absorption and light scattering
ANALYTICAL
BIOCHEMISTRY
201,9‘i-98
(19%)
The Structure of Glutaraldehyde in Aqueous Solution Determined by Ultraviolet Absorption and Light Scattering Jun-ichi Kawahara,’ National
Received
Chemical
August
Takao Ohmori, Teiji Ohkubo, Shigeru Hattori, and Mitsutaka Kawamura
Laboratory
for Industry,
Tsukuba,
Ibaraki
305, Japan
16,199l
However, commercial GA is supplied in and the cross-linking reaction with proteins is carried out in aqueous solution, and GA reacts with water in various ways. Thus there is a considerable problem with the fact that Monsan et al. analyzed the molecular structure of aqueous GA itself only in organic solvents (tetrahydrofuran in gel chromatography, chloroform/acetone in thin-layer chromatography, and deuterated chloroform or carbon tetrachloride in NMR). Furthermore, in anhydrous solvents the equilibrium between monomeric and polymerized GA possibly shifts to the latter, which produces water (Fig. 1). Other studies have similar fundamental problems (l3,5), except that of Korn et al. (4). Some researchers conducted experiments in D,O (1,2). However, since an exchange of deuterium for hydrogen bound to a-carbon might occur (4), it may give erroneous results to compare the peak intensities of H-NMR. Moreover, the hydration equilibrium constants for monoaldehydes are Glutaraldehyde (GA)2 has been widely used in cross- reported to differ in Hz0 and D,O (7), and this will problinking proteins, fixing tissue samples, etc. Although the ably also be the case with GA. Whipple and Ruta (3) measured 13C-NMR, but it is known that direct comparistructure and the cross-linking mechanism of the.crosslinking reagents are of prime importance on their use, son of the peak intensities is not quantitative in 13CNMR (8). the actual structure that GA takes in aqueous solution In the present study, the molecular structure of GA in has been, unlike those of the other cross-linking reaqueous solutions was directly investigated with uv abagents, the subject of much debate (l-5). At present, it sorption and light scattering. It was found that aqueous seemsthat the structure proposed by Monsan et al. (5) is GA consists mainly of cyclic hemiacetal structure and accepted (6). They proposed that commercial aqueous GA consists mainly of polymeric species with an (Y,@- does not contain any c@-unsaturated structure. It was unsaturated structure at neutral or slightly basic pH also found that the relative abundances of monomeric and polymeric species vary markedly according to the and that these were the molecular species that produced GA concentration. the cross-linking reaction with proteins.
The structure of glutaraldehyde (GA) in aqueous solutions has been the subject of much debate. Since there were fundamental problems in the experiments in the preceding studies, in this article, the structure of GA was investigated with uv absorption and light scattering to avoid those problems. It was discovered that 70% glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution, the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. cY&Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde. 0 1992 Academic Press, Inc.
MATERIALS ’ To whom correspondence should be addressed. * Abbreviations used: GA, glutaraldehyde; I&, weight-average lecular weight.
mo-
AND
METHODS
Commercial 70% (w/v) and ca. 20% (w/v) GA in aqueous solution (EM grade, purchased from Wako, pH
94 All
Copyright 0 1992 rights of reproduction
0003-2697/92 $3.00 by Academic Press, Inc. in any form reserved.
ULTRAVIOLET
INVESTIGATION
OF
GLUTARALDEHYDE
95
STRUCTURE
tonaldehyde, due to the resonance interaction with the 0
PI-
+H,O
CHO CHO (1)
(IV)
-Hz0
+H,O G n n CHO CH(OH), U-QHC m (II)
FHO fCH0 CH2-(CH2)2-CH C-(CHJ2-CH
CWW,
{PO C-(CH2)2-CH0
WI) FIG. 1. Possible and the reaction
molecular structures paths between them.
of GA in aqueous
solutions
3.5-4.0) were used as samples without purification. Ultraviolet absorption of GA was measured with a Beckman DU-70 spectrophotometer equipped with a thermostating circulator, in cuvettes of 10 or 2 mm in path length. Light scattering was measured at 25°C with an Otsuka DLS-700s light scattering photometer at 633 nm, calibrated with benzene (9). The optical clarification was performed with Teflon filters. The specific refractive index increment (dnldc) was obtained with a Chromatix KMX-16 refractometer at the same wavelength, calibrated with NaCl solution. The deoxygenation of redistilled water, which was used to dilute the GA solution, was performed by replacing the gaseous oxygen in the water with gaseous nitrogen.
ethylenic double bond (Fig. 2b). The strong absorption of crotonaldehyde at 223.5 nm is assigned to the ?T--?T* allowed transition of the ethylenic double bond, which is in conjugation with an aldehyde group (Fig. 2~). Seventy percent GA solution, even if it is pure, contains a considerable amount of glassy structures. When this solution is diluted, although white turbidity appears temporarily, it soon becomes clear again, with a gradual change in absorption spectrum (Fig. 2g and Fig. 3A). The main peak indicates the existence of a free aldehyde group, which has n-?r* carbonyl absorption band at ca. 280 nm, as seen in Fig. 2a. However, the apparent extinction coefficient of the aldehyde peak is much lower than that of n-butyraldehyde, in the cases of both 70% solution (extrapolated to the zero time in Fig. 3A) and 0.4% solution (extrapolated to the infinite time in Fig. 3A). This fact indicates that, although large portions of the aldehyde groups in aliphatic monoaldehydes are free in aqueous solutions (7,10-12), only a very small portion is free in the case of GA in aqueous solution. Since the hydration equilibrium constant for GA is not expected to be very different from those for aliphatic monoaldehydes, the low abundance of the free aldehyde group in GA indicates that the structures IV or V are predominant in aqueous GA solution (discussed in detail later). Moreover, the spectral change in the dilution process indicates the difference in (the abundances of) the molecular structures of GA between 70% and dilute solutions (described later). The shape of the absorption spectrum of 20% GA solution was almost the same as that of more dilute GA solution (data not shown) and underwent only a minor
0.25 1"
RESULTS AND DISCUSSION
Figure 1 shows the possible molecular structures of GA and includes all of the structures that have been referred to in previous studies (l-5). We also propose here the most probable reaction paths deduced from the properties of aldehydes; there have been some discrepancies also in the reaction paths that connect them. VI is the average structure of the unsaturated polymerized GA. (The number of methylene groups between the neighboring ethylenic double bonds might be 1,2, or 3, but the weighted average is exactly equal to 2.) VI might also contain ring structures at the end of its main chain. The pendent aldehyde groups of VI would be scarcely hydrated since the carbonyl form is stabilized by conjugation (10). Figure 2 shows the uv absorption spectra of some aldehydes and GA in its dilution process. The wavelength of maximum absorption is shifted and the extinction coefficient is increased for the aldehyde group in cro-
0.2 OJ 0 z 0.15 a L =: 0.1 s 0.05 I
O 210
230
250
Wave1 ength FIG. 2.
270
290
310
(nml
Ultraviolet absorption spectra of GA and its related compounds in H,O: (a) 16 mM n-butyraldehyde. (b) 5 mM crotonaldehyde. (c) 0.01 mM crotonaldehyde. (g) 70% commercial GA, preincubated at 2O”C, was diluted with deoxygenated water to 0.4% concentration (40 mM) and measured at 2, 7, 12, 17, and 22 min after dilution with increasing absorbance. The temperature was kept at 20°C after dilution also.
96
KAWAHARA
o-24o-
I
$ 0.140' z 0.200 s 0.195
B
, 0.19ob
, 50
100
150
200
I 250
Ti me (mi n I FIG. 3.
The absorbance variation of GA at 280 nm after dilution. (A) The condition is the same as that for Fig. 2g, except that the absorbance is plotted against the time after dilution. (B) The condition is the same as that for (A), except that ca. 20% commercial GA was used as starting solution.
change in the dilution process (Fig. 3B). This indicates that the structure of GA in 20% solution is similar to that in more dilute solution. The comparison of the absorption spectra of GA with those of crotonaldehyde (Figs. 2b and 2c) indicates that the unsaturated structure scarcely exists over a wide range of GA concentration, or in the transient state of the dilution process. Although a small shoulder is observed at ca. 235 nm in the spectra of GA, and even if it is assumed to be due to the existence of the conjugated unsaturated structure, the content would be far below 0.1% by weight, since crotonaldehyde (Fig. 2c) and similar structures have strong absorption near this wavelength. (The a---** transition of the ethylenic double bond in structure VI is expected to produce absorption with similar intensity but at a wavelength ca. 10 nm longer than that of crotonaldehyde, since an extra hydrocarbon chain is attached to a-carbon in the case of VI (13)). It should be noted, however, that some commercial GA samples do contain impurities with significant absorption at 235 nm (data not shown). The molecular weight of GA in aqueous solution was determined with light scattering. Figure 4 shows the Zimm plot (14) for GA in H,O. Kc/R, values were doubly extrapolated to 8 = 0 and c = 0 by straight lines to obtain (Kc/R,), (plotted as a,). This shows that the weightaverage molecular weight (M,) of GA, which is equal to the reciprocal of (Kc/R,),, is ca. 100. Since i& is very sensitive to the existence of high-molecular-weight species, this clearly indicates that GA is almost monomeric in 2-7% aqueous solution. In light scattering measure-
ET AL.
ments, we neglected the correction for isothermal compressibility of the solution (15); this would cause errors of only a few percent and does not seem to affect our conclusions. The pH effect on the molecular structure of GA, which was reported previously (2,5), was also examined with light scattering (one concentration method (16)). Figure 5 shows that M, of GA is ca. 100 at pH 8.0. The scattering pattern did not change for at least 2 days at 25’C. The same results were obtained for pH 7.0 and 6.0. Moreover, M.,, of GA in acidic solutions was also shown to be ca. 100 (Fig. 6). M, did not change for more than a week despite the heat treatment. These facts indicate that, contrary to earlier reports, dilute GA is almost monomeric not only in acidic solutions but also in neutral and slightly basic solutions for an extended period of time. When the results obtained from uv absorption and light scattering are combined, it seems probable that most of the GA takes the structure of IV and the amount of V is negligible in dilute solution. On the other hand, the gradual increase of absorption at 280 nm in the dilution process of 70% GA indicates that 70% aqueous GA contains a considerable amount of V structure, which converts to monomer in the dilution process. Cross-linking reaction with proteins is usually carried out in less than 1% GA concentration. Under such conditions, considering the effect of GA concentration on the equilibrium between the monomeric and the polymeric states, the concentration of polymeric GA is expected to be even lower than that in the present study. Since the conversion of V to monomers is rather slow (Fig. 3A), more attention should be paid to the time after dilution, when quantitative results are necessary (for example, in the case of tissue fixation, where the control of the osmotic pressure is important).
1.4
-
& z 29 1.2 e s
-
-
-
1.0
I -0.8
-0.4
0
0.4
0.8
Sin* (e/21-1Oc FIG. 4. The Zimm plot for GA in H,O. The concentrations are 1.78 (a), 3.62 (b), 5.25 (c), and 7.03% (d) (w/v), with a dnldc value of 0.175 ml - g-l. Each concentration was prepared by diluting 70% commercial GA with redistilled water several hours before measurements.
ULTRAVIOLET
INVESTIGATION
OF
1.1I’
1: *0
0.4
0.8
si n2 (B/2)
FIG. 5. The molecular weight of GA in neutral or slightly basic aqueous solutions: 70% commercial GA was diluted to 3.4% (w/v) concentration with redistilled water and stored at room temperature for several hours. The solution was then mixed with the same volume of 200 mM sodium phosphate buffer to pH 8.0. The light scattering measurement was performed just after (A), 30 min after (B), and 60 min after (C) the pH adjustment.
In aqueous solutions the possible interactions between the free or hydrated aldehyde groups in the same GA molecule are expected to be mostly suppressed by the interactions (hydrogen bond formation, electrostatic interaction) between each aldehyde group and water molecules, except the formation of cyclic hemiacetal structure. Under such conditions, we may treat the aldehyde groups of the GA molecule as independent of each other when we consider the extinction coefficient and the equilibria among I, II, and III. With this hypothesis the relative amount of each structure (in monomer units) in aqueous solutions can be estimated. Since c- of the free (not hydrated) aldehyde group of acetaldehyde, propionaldehyde, n-butyraldehyde, and ibutyraldehyde were shown to have similar values with X, of ca. 280 nm in H,O (7), c280nm of the free aldehyde group of GA is also expected to have a comparable value. The extinction coefficient of the hydrated aldehyde is virtually zero at this wavelength (7). Thus, from the absorbance A = 0.220 (obtained from the extrapolation to the infinite time in Fig. 3A), ca. 16% of the aldehyde groups are estimated to be free in dilute solution at 20°C. Moreover, the equilibrium constant for hydration (K,,) does not depend strongly on the aliphatic chain length in monoaldehydes (7). Therefore, if we use Ki., of
GLUTARALDEHYDE
97
STRUCTURE
n-butyraldehyde (0.48 (7)) for that of GA and assume the concentration of I to be X, then the concentrations of II and III are estimated to be 2x& and 2xK2,, respectively. Since the concentration of the aldehyde group is (2x + 2x&) and is equal to twice 16% of the total GA concentration (in monomer units), we obtain the values 10.8, 10.4, and 2.5% of the total GA concentration (in monomer units) for the concentrations of I, II, and III, respectively. Hence, the relative amounts of I, II, III, IV, and V in dilute GA solution would not be very different from 11, 10,2.5,76, and 0% (in monomer units), respectively. (The content of V is estimated to be virtually zero from the result of light scattering. See Figs. 4,5, and 6.) On the other hand, in 70% GA solution, about 11% of aldehyde group is estimated to be free at 20°C from the absorbance A = 0.1486 (obtained from extrapolation to zero time in Fig. 3A). If we use the same Kh value as that in dilute GA solution, values of 7.5, 6.8, and 1.7% are obtained for the concentrations of I, II, and III, respectively, through the calculation mentioned above. However, if we consider the fact that the concentration of water is significantly lower in this case, the equilibria between I, II, and III are expected to be considerably shifted to the left in Fig. 1, and the amounts are expected to be between 7.5 and 11% (probably nearly ll%), less than 6.8% (probably considerably less than 6.8%), and less than 1.7% (probably nearly O%), respectively. Since the increase of absorbance from 0.1486 to 0.220 is considered to be caused by the conversion of the whole V to IV but is expected to be canceled considerably by the shifts of equilibria to the right in Fig. 1 in-
120
s
100 * ; “pi I 80 c
0
f
I
I
I
I
2
4
6
8
10
Ti me (d 1 FIG. 6. The molecular weight of GA in acidic aqueous solutions. The conditions are the same as those given in the legend to Fig. 5 except the adjusted pH. M, was plotted against the time after the pH adjustment to 5.0 (O), 4.0 (A), and 3.0 (X), respectively. At the point indicated by the arrow, the temperature of the three sample solutions was elevated to 60°C for 1 h.
98
KAWAHARA
duced by the significant increase of water activity in the dilution process, the amount of V is expected to be considerably larger than 32.5% (l-0.1486/0.220 = 0.325). The rest takes structure IV. It seems that, in 70% GA solution, not only the high concentration of GA but also the low concentration of water play an important role in the formation of V and in other shifts of equilibria from the state of dilute solutions. Korn et al., who also studied the 70% GA solution, suggested a value of 15% for I and 85% for (IV + V) at 25”C, on the basis of their H-NMR experiments (4). (They also suggested the possibility of a small amount of II and III). Considering the effect of temperature on the equilibria between molecular structures of GA (4), their results suggest that the expected amount of I is less than 15% at 20°C. Since the sample used by Korn et al. is unlikely to have had exactly the same composition as that used here (the actual concentration of commercial 70% GA solution is not necessarily 70%, and this deviation of the concentration can cause a slight variation of the composition), the reported values are in good agreement with the present study. For those using GA as a cross-linking reagent for proteins, the following comments might be useful. It is well known that GA cross-links proteins very effectively. As a matter of fact, this monomeric GA (diluted from 70% commercial GA and stored at room temperature for several hours) cross-links myosin molecules to form a gel in GA concentrations as low as 0.01% (data not shown). It might seem rather difficult to explain this effectiveness of GA in cross-linking reactions, if principal species of GA in aqueous solution are monomers. Our results, however, do not necessarily indicate that GA cross-links proteins in the monomeric form. Rather, there is a possibility that polymeric structures like VI are involved in the actual cross-linking reactions, since in the reaction of GA with a trace amount of n-amylamine, which is considered an analogue of the side chain of Lys residue, a significant absorption peak that closely resembles that expected for the x--x* transition of the ethylenic double bond in the structure VI is produced (data not shown). In other words, it could be that monomeric GA is converted to polymeric forms by the action of amino group, and this newly produced polymeric GA plays a major role in the cross-linking reaction of proteins. This cross-linking mechanism of GA is the next important problem to be elucidated and now is under investigation in our laboratory.
ET
AL.
CONCLUSION
The structure of glutaraldehyde in aqueous solutions was investigated with uv absorption and light scattering. Seventy percent glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. a&Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde. ACKNOWLEDGMENT The authors thank Drs. Hideo Orita, Masao Shimizu, Akiko Takatsu, Masaaki Sugiura, and Arnfinn Andersen for their helpful discussion. This work was supported in part by special coordination funds for promoting science and technology from the Science and Technology Agency, Japan.
REFERENCES 1. Richards, 233.
F. M.,
and Knowles,
J. R. (1968)
J. Mol.
Biol.
37,231-
2. Hardy, P. M., Nicholls, Commun., 565-566.
A. C., and Rydon,
3. Whipple, E. B., and Ruta, 4. Korn, A. H., Feairheller, Mol. Biol. 65,525-529.
M. (1974) J. Org. Chem. 39,1666-1668. S. H., and Filachione, E. M. (1972)
5. Monsan, P., Puzo, G., and Mazarguil, 1281-1292. 6. Peters, K., and Richards, F. M. (1977) 523-551.
H. N. (1969)
H. (1975) Ann.
&em.
J.
Biochimie
57,
Reu. Biochem.
46,
7. Gruen, L. C., and McTigue, P. T. (1963) J. Chem. Sot., 5217-5223. 8. Silverstein, R. M., Bassler, G. C., and Morrill, T. C. (1981) Spectrometric Identification of Organic Compounds, 4th ed., pp. 249303, Wiley, New York. 9. Pike, &em.
E. R., Pomeroy, W. R. M., Phys. 62,3188-3192.
and Vaughan,
J. M.
10. Hooper, D. L. (1967) J. Chem. Sot. B, 169-170. P., Luz, Z., and Samuel, D. (1967) J. Am. 11. Greenzaid, 89, 749-756. 12. Bell, R. P. (1966) Adu. Phys. Org. Chem. 4,1-27. 13. Fieser, L. M., Phenanthrene,
and Fieser, M. (1949) p. 184 ff., Reinhold,
Natural Products New York.
(1975)
Chem.
Related
J.
Sot.
to
14. Zimm, B. H. (1948) J. Chem. Phys. 16, 1099-1116. T., Nakahara, H., and Hattori, S. (1977) Bull. Chem. 15. Kamata, Sot. Jpn. 60,2558-2563. 16. Kamata, T., and Nakabara, H. (1973) J. Colloid Znterface Sci. 43, 89-96.
BIOCHEMISTRY
201,9‘i-98
(19%)
The Structure of Glutaraldehyde in Aqueous Solution Determined by Ultraviolet Absorption and Light Scattering Jun-ichi Kawahara,’ National
Received
Chemical
August
Takao Ohmori, Teiji Ohkubo, Shigeru Hattori, and Mitsutaka Kawamura
Laboratory
for Industry,
Tsukuba,
Ibaraki
305, Japan
16,199l
However, commercial GA is supplied in and the cross-linking reaction with proteins is carried out in aqueous solution, and GA reacts with water in various ways. Thus there is a considerable problem with the fact that Monsan et al. analyzed the molecular structure of aqueous GA itself only in organic solvents (tetrahydrofuran in gel chromatography, chloroform/acetone in thin-layer chromatography, and deuterated chloroform or carbon tetrachloride in NMR). Furthermore, in anhydrous solvents the equilibrium between monomeric and polymerized GA possibly shifts to the latter, which produces water (Fig. 1). Other studies have similar fundamental problems (l3,5), except that of Korn et al. (4). Some researchers conducted experiments in D,O (1,2). However, since an exchange of deuterium for hydrogen bound to a-carbon might occur (4), it may give erroneous results to compare the peak intensities of H-NMR. Moreover, the hydration equilibrium constants for monoaldehydes are Glutaraldehyde (GA)2 has been widely used in cross- reported to differ in Hz0 and D,O (7), and this will problinking proteins, fixing tissue samples, etc. Although the ably also be the case with GA. Whipple and Ruta (3) measured 13C-NMR, but it is known that direct comparistructure and the cross-linking mechanism of the.crosslinking reagents are of prime importance on their use, son of the peak intensities is not quantitative in 13CNMR (8). the actual structure that GA takes in aqueous solution In the present study, the molecular structure of GA in has been, unlike those of the other cross-linking reaqueous solutions was directly investigated with uv abagents, the subject of much debate (l-5). At present, it sorption and light scattering. It was found that aqueous seemsthat the structure proposed by Monsan et al. (5) is GA consists mainly of cyclic hemiacetal structure and accepted (6). They proposed that commercial aqueous GA consists mainly of polymeric species with an (Y,@- does not contain any c@-unsaturated structure. It was unsaturated structure at neutral or slightly basic pH also found that the relative abundances of monomeric and polymeric species vary markedly according to the and that these were the molecular species that produced GA concentration. the cross-linking reaction with proteins.
The structure of glutaraldehyde (GA) in aqueous solutions has been the subject of much debate. Since there were fundamental problems in the experiments in the preceding studies, in this article, the structure of GA was investigated with uv absorption and light scattering to avoid those problems. It was discovered that 70% glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution, the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. cY&Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde. 0 1992 Academic Press, Inc.
MATERIALS ’ To whom correspondence should be addressed. * Abbreviations used: GA, glutaraldehyde; I&, weight-average lecular weight.
mo-
AND
METHODS
Commercial 70% (w/v) and ca. 20% (w/v) GA in aqueous solution (EM grade, purchased from Wako, pH
94 All
Copyright 0 1992 rights of reproduction
0003-2697/92 $3.00 by Academic Press, Inc. in any form reserved.
ULTRAVIOLET
INVESTIGATION
OF
GLUTARALDEHYDE
95
STRUCTURE
tonaldehyde, due to the resonance interaction with the 0
PI-
+H,O
CHO CHO (1)
(IV)
-Hz0
+H,O G n n CHO CH(OH), U-QHC m (II)
FHO fCH0 CH2-(CH2)2-CH C-(CHJ2-CH
CWW,
{PO C-(CH2)2-CH0
WI) FIG. 1. Possible and the reaction
molecular structures paths between them.
of GA in aqueous
solutions
3.5-4.0) were used as samples without purification. Ultraviolet absorption of GA was measured with a Beckman DU-70 spectrophotometer equipped with a thermostating circulator, in cuvettes of 10 or 2 mm in path length. Light scattering was measured at 25°C with an Otsuka DLS-700s light scattering photometer at 633 nm, calibrated with benzene (9). The optical clarification was performed with Teflon filters. The specific refractive index increment (dnldc) was obtained with a Chromatix KMX-16 refractometer at the same wavelength, calibrated with NaCl solution. The deoxygenation of redistilled water, which was used to dilute the GA solution, was performed by replacing the gaseous oxygen in the water with gaseous nitrogen.
ethylenic double bond (Fig. 2b). The strong absorption of crotonaldehyde at 223.5 nm is assigned to the ?T--?T* allowed transition of the ethylenic double bond, which is in conjugation with an aldehyde group (Fig. 2~). Seventy percent GA solution, even if it is pure, contains a considerable amount of glassy structures. When this solution is diluted, although white turbidity appears temporarily, it soon becomes clear again, with a gradual change in absorption spectrum (Fig. 2g and Fig. 3A). The main peak indicates the existence of a free aldehyde group, which has n-?r* carbonyl absorption band at ca. 280 nm, as seen in Fig. 2a. However, the apparent extinction coefficient of the aldehyde peak is much lower than that of n-butyraldehyde, in the cases of both 70% solution (extrapolated to the zero time in Fig. 3A) and 0.4% solution (extrapolated to the infinite time in Fig. 3A). This fact indicates that, although large portions of the aldehyde groups in aliphatic monoaldehydes are free in aqueous solutions (7,10-12), only a very small portion is free in the case of GA in aqueous solution. Since the hydration equilibrium constant for GA is not expected to be very different from those for aliphatic monoaldehydes, the low abundance of the free aldehyde group in GA indicates that the structures IV or V are predominant in aqueous GA solution (discussed in detail later). Moreover, the spectral change in the dilution process indicates the difference in (the abundances of) the molecular structures of GA between 70% and dilute solutions (described later). The shape of the absorption spectrum of 20% GA solution was almost the same as that of more dilute GA solution (data not shown) and underwent only a minor
0.25 1"
RESULTS AND DISCUSSION
Figure 1 shows the possible molecular structures of GA and includes all of the structures that have been referred to in previous studies (l-5). We also propose here the most probable reaction paths deduced from the properties of aldehydes; there have been some discrepancies also in the reaction paths that connect them. VI is the average structure of the unsaturated polymerized GA. (The number of methylene groups between the neighboring ethylenic double bonds might be 1,2, or 3, but the weighted average is exactly equal to 2.) VI might also contain ring structures at the end of its main chain. The pendent aldehyde groups of VI would be scarcely hydrated since the carbonyl form is stabilized by conjugation (10). Figure 2 shows the uv absorption spectra of some aldehydes and GA in its dilution process. The wavelength of maximum absorption is shifted and the extinction coefficient is increased for the aldehyde group in cro-
0.2 OJ 0 z 0.15 a L =: 0.1 s 0.05 I
O 210
230
250
Wave1 ength FIG. 2.
270
290
310
(nml
Ultraviolet absorption spectra of GA and its related compounds in H,O: (a) 16 mM n-butyraldehyde. (b) 5 mM crotonaldehyde. (c) 0.01 mM crotonaldehyde. (g) 70% commercial GA, preincubated at 2O”C, was diluted with deoxygenated water to 0.4% concentration (40 mM) and measured at 2, 7, 12, 17, and 22 min after dilution with increasing absorbance. The temperature was kept at 20°C after dilution also.
96
KAWAHARA
o-24o-
I
$ 0.140' z 0.200 s 0.195
B
, 0.19ob
, 50
100
150
200
I 250
Ti me (mi n I FIG. 3.
The absorbance variation of GA at 280 nm after dilution. (A) The condition is the same as that for Fig. 2g, except that the absorbance is plotted against the time after dilution. (B) The condition is the same as that for (A), except that ca. 20% commercial GA was used as starting solution.
change in the dilution process (Fig. 3B). This indicates that the structure of GA in 20% solution is similar to that in more dilute solution. The comparison of the absorption spectra of GA with those of crotonaldehyde (Figs. 2b and 2c) indicates that the unsaturated structure scarcely exists over a wide range of GA concentration, or in the transient state of the dilution process. Although a small shoulder is observed at ca. 235 nm in the spectra of GA, and even if it is assumed to be due to the existence of the conjugated unsaturated structure, the content would be far below 0.1% by weight, since crotonaldehyde (Fig. 2c) and similar structures have strong absorption near this wavelength. (The a---** transition of the ethylenic double bond in structure VI is expected to produce absorption with similar intensity but at a wavelength ca. 10 nm longer than that of crotonaldehyde, since an extra hydrocarbon chain is attached to a-carbon in the case of VI (13)). It should be noted, however, that some commercial GA samples do contain impurities with significant absorption at 235 nm (data not shown). The molecular weight of GA in aqueous solution was determined with light scattering. Figure 4 shows the Zimm plot (14) for GA in H,O. Kc/R, values were doubly extrapolated to 8 = 0 and c = 0 by straight lines to obtain (Kc/R,), (plotted as a,). This shows that the weightaverage molecular weight (M,) of GA, which is equal to the reciprocal of (Kc/R,),, is ca. 100. Since i& is very sensitive to the existence of high-molecular-weight species, this clearly indicates that GA is almost monomeric in 2-7% aqueous solution. In light scattering measure-
ET AL.
ments, we neglected the correction for isothermal compressibility of the solution (15); this would cause errors of only a few percent and does not seem to affect our conclusions. The pH effect on the molecular structure of GA, which was reported previously (2,5), was also examined with light scattering (one concentration method (16)). Figure 5 shows that M, of GA is ca. 100 at pH 8.0. The scattering pattern did not change for at least 2 days at 25’C. The same results were obtained for pH 7.0 and 6.0. Moreover, M.,, of GA in acidic solutions was also shown to be ca. 100 (Fig. 6). M, did not change for more than a week despite the heat treatment. These facts indicate that, contrary to earlier reports, dilute GA is almost monomeric not only in acidic solutions but also in neutral and slightly basic solutions for an extended period of time. When the results obtained from uv absorption and light scattering are combined, it seems probable that most of the GA takes the structure of IV and the amount of V is negligible in dilute solution. On the other hand, the gradual increase of absorption at 280 nm in the dilution process of 70% GA indicates that 70% aqueous GA contains a considerable amount of V structure, which converts to monomer in the dilution process. Cross-linking reaction with proteins is usually carried out in less than 1% GA concentration. Under such conditions, considering the effect of GA concentration on the equilibrium between the monomeric and the polymeric states, the concentration of polymeric GA is expected to be even lower than that in the present study. Since the conversion of V to monomers is rather slow (Fig. 3A), more attention should be paid to the time after dilution, when quantitative results are necessary (for example, in the case of tissue fixation, where the control of the osmotic pressure is important).
1.4
-
& z 29 1.2 e s
-
-
-
1.0
I -0.8
-0.4
0
0.4
0.8
Sin* (e/21-1Oc FIG. 4. The Zimm plot for GA in H,O. The concentrations are 1.78 (a), 3.62 (b), 5.25 (c), and 7.03% (d) (w/v), with a dnldc value of 0.175 ml - g-l. Each concentration was prepared by diluting 70% commercial GA with redistilled water several hours before measurements.
ULTRAVIOLET
INVESTIGATION
OF
1.1I’
1: *0
0.4
0.8
si n2 (B/2)
FIG. 5. The molecular weight of GA in neutral or slightly basic aqueous solutions: 70% commercial GA was diluted to 3.4% (w/v) concentration with redistilled water and stored at room temperature for several hours. The solution was then mixed with the same volume of 200 mM sodium phosphate buffer to pH 8.0. The light scattering measurement was performed just after (A), 30 min after (B), and 60 min after (C) the pH adjustment.
In aqueous solutions the possible interactions between the free or hydrated aldehyde groups in the same GA molecule are expected to be mostly suppressed by the interactions (hydrogen bond formation, electrostatic interaction) between each aldehyde group and water molecules, except the formation of cyclic hemiacetal structure. Under such conditions, we may treat the aldehyde groups of the GA molecule as independent of each other when we consider the extinction coefficient and the equilibria among I, II, and III. With this hypothesis the relative amount of each structure (in monomer units) in aqueous solutions can be estimated. Since c- of the free (not hydrated) aldehyde group of acetaldehyde, propionaldehyde, n-butyraldehyde, and ibutyraldehyde were shown to have similar values with X, of ca. 280 nm in H,O (7), c280nm of the free aldehyde group of GA is also expected to have a comparable value. The extinction coefficient of the hydrated aldehyde is virtually zero at this wavelength (7). Thus, from the absorbance A = 0.220 (obtained from the extrapolation to the infinite time in Fig. 3A), ca. 16% of the aldehyde groups are estimated to be free in dilute solution at 20°C. Moreover, the equilibrium constant for hydration (K,,) does not depend strongly on the aliphatic chain length in monoaldehydes (7). Therefore, if we use Ki., of
GLUTARALDEHYDE
97
STRUCTURE
n-butyraldehyde (0.48 (7)) for that of GA and assume the concentration of I to be X, then the concentrations of II and III are estimated to be 2x& and 2xK2,, respectively. Since the concentration of the aldehyde group is (2x + 2x&) and is equal to twice 16% of the total GA concentration (in monomer units), we obtain the values 10.8, 10.4, and 2.5% of the total GA concentration (in monomer units) for the concentrations of I, II, and III, respectively. Hence, the relative amounts of I, II, III, IV, and V in dilute GA solution would not be very different from 11, 10,2.5,76, and 0% (in monomer units), respectively. (The content of V is estimated to be virtually zero from the result of light scattering. See Figs. 4,5, and 6.) On the other hand, in 70% GA solution, about 11% of aldehyde group is estimated to be free at 20°C from the absorbance A = 0.1486 (obtained from extrapolation to zero time in Fig. 3A). If we use the same Kh value as that in dilute GA solution, values of 7.5, 6.8, and 1.7% are obtained for the concentrations of I, II, and III, respectively, through the calculation mentioned above. However, if we consider the fact that the concentration of water is significantly lower in this case, the equilibria between I, II, and III are expected to be considerably shifted to the left in Fig. 1, and the amounts are expected to be between 7.5 and 11% (probably nearly ll%), less than 6.8% (probably considerably less than 6.8%), and less than 1.7% (probably nearly O%), respectively. Since the increase of absorbance from 0.1486 to 0.220 is considered to be caused by the conversion of the whole V to IV but is expected to be canceled considerably by the shifts of equilibria to the right in Fig. 1 in-
120
s
100 * ; “pi I 80 c
0
f
I
I
I
I
2
4
6
8
10
Ti me (d 1 FIG. 6. The molecular weight of GA in acidic aqueous solutions. The conditions are the same as those given in the legend to Fig. 5 except the adjusted pH. M, was plotted against the time after the pH adjustment to 5.0 (O), 4.0 (A), and 3.0 (X), respectively. At the point indicated by the arrow, the temperature of the three sample solutions was elevated to 60°C for 1 h.
98
KAWAHARA
duced by the significant increase of water activity in the dilution process, the amount of V is expected to be considerably larger than 32.5% (l-0.1486/0.220 = 0.325). The rest takes structure IV. It seems that, in 70% GA solution, not only the high concentration of GA but also the low concentration of water play an important role in the formation of V and in other shifts of equilibria from the state of dilute solutions. Korn et al., who also studied the 70% GA solution, suggested a value of 15% for I and 85% for (IV + V) at 25”C, on the basis of their H-NMR experiments (4). (They also suggested the possibility of a small amount of II and III). Considering the effect of temperature on the equilibria between molecular structures of GA (4), their results suggest that the expected amount of I is less than 15% at 20°C. Since the sample used by Korn et al. is unlikely to have had exactly the same composition as that used here (the actual concentration of commercial 70% GA solution is not necessarily 70%, and this deviation of the concentration can cause a slight variation of the composition), the reported values are in good agreement with the present study. For those using GA as a cross-linking reagent for proteins, the following comments might be useful. It is well known that GA cross-links proteins very effectively. As a matter of fact, this monomeric GA (diluted from 70% commercial GA and stored at room temperature for several hours) cross-links myosin molecules to form a gel in GA concentrations as low as 0.01% (data not shown). It might seem rather difficult to explain this effectiveness of GA in cross-linking reactions, if principal species of GA in aqueous solution are monomers. Our results, however, do not necessarily indicate that GA cross-links proteins in the monomeric form. Rather, there is a possibility that polymeric structures like VI are involved in the actual cross-linking reactions, since in the reaction of GA with a trace amount of n-amylamine, which is considered an analogue of the side chain of Lys residue, a significant absorption peak that closely resembles that expected for the x--x* transition of the ethylenic double bond in the structure VI is produced (data not shown). In other words, it could be that monomeric GA is converted to polymeric forms by the action of amino group, and this newly produced polymeric GA plays a major role in the cross-linking reaction of proteins. This cross-linking mechanism of GA is the next important problem to be elucidated and now is under investigation in our laboratory.
ET
AL.
CONCLUSION
The structure of glutaraldehyde in aqueous solutions was investigated with uv absorption and light scattering. Seventy percent glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. a&Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde. ACKNOWLEDGMENT The authors thank Drs. Hideo Orita, Masao Shimizu, Akiko Takatsu, Masaaki Sugiura, and Arnfinn Andersen for their helpful discussion. This work was supported in part by special coordination funds for promoting science and technology from the Science and Technology Agency, Japan.
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