ANALYTICAL BIOCHEMISTRY 201,9‘i-98 (19%)

The Structure of Glutaraldehyde in Aqueous Solution

Determined by Ultraviolet Absorption
and Light Scattering

Jun-ichi Kawahara,’ Takao Ohmori, Teiji Ohkubo, Shigeru Hattori, and Mitsutaka Kawamura
National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305, Japan

Received August 16,199l

However, commercial GA is supplied in and the

The structure of glutaraldehyde (GA) in aqueous so-
cross-linking reaction with proteins is carried out in
lutions has been the subject of much debate. Since there aqueous solution, and GA reacts with water in various
were fundamental problems ways. Thus there is a considerable problem with the fact
in the experiments in the
preceding studies, in this article, that Monsan et al. analyzed the molecular structure of
the structure of GA
was investigated with uv absorption and light scatter-
aqueous GA itself only in organic solvents (tetrahydro-
ing to avoid those problems. It was discovered that 70%
furan in gel chromatography, chloroform/acetone in
glutaraldehyde solution contains a large quantity of
thin-layer chromatography, and deuterated chloroform
polymeric species with cyclic hemiacetal structure. On
or carbon tetrachloride in NMR). Furthermore, in an-
dilution, the polymerized glutaraldehyde slowly con-
hydrous solvents the equilibrium between monomeric
verted to monomers. In dilute solution, glutaraldehyde
and polymerized GA possibly shifts to the latter, which
is almost monomeric at pH 3-8, the major portion tak-
produces water (Fig. 1).
ing the cyclic hemiacetal structure. The structure of GA
Other studies have similar fundamental problems (l-
in 20% solution is similar to that in more dilute solu-
3,5), except that of Korn et al. (4). Some researchers
tion. cY&Unsaturated structure does not exist in
aqueous solution regardless conducted experiments in D,O (1,2). However, since an
of the concentration of glu-
taraldehyde. 0 1992 Academic Press, Inc.
exchange of deuterium for hydrogen bound to a-carbon
might occur (4), it may give erroneous results to com-
pare the peak intensities of H-NMR. Moreover, the hy-
dration 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 prob-
linking proteins, fixing tissue samples, etc. Although the ably also be the case with GA. Whipple and Ruta (3)
structure and the cross-linking mechanism of the.cross- measured 13C-NMR, but it is known that direct compari-
linking reagents are of prime importance on their use, son of the peak intensities is not quantitative in 13C-
the actual structure that GA takes in aqueous solution NMR (8).
has been, unlike those of the other cross-linking re- In the present study, the molecular structure of GA in
agents, the subject of much debate (l-5). At present, it aqueous solutions was directly investigated with uv ab-
seemsthat the structure proposed by Monsan et al. (5) is sorption and light scattering. It was found that aqueous
accepted (6). They proposed that commercial aqueous GA consists mainly of cyclic hemiacetal structure and
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 that these were the molecular species that produced and polymeric species vary markedly according to the
the cross-linking reaction with proteins. GA concentration.

MATERIALS AND METHODS

’ To whom correspondence should be addressed.
* Abbreviations used: GA, glutaraldehyde; I&, weight-average mo- Commercial 70% (w/v) and ca. 20% (w/v) GA in
lecular weight. aqueous solution (EM grade, purchased from Wako, pH
94 0003-2697/92 $3.00
Copyright 0 1992 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 ULTRAVIOLET INVESTIGATION OF GLUTARALDEHYDE STRUCTURE 95

tonaldehyde, due to the resonance interaction with the

ethylenic double bond (Fig. 2b). The strong absorption
0 of crotonaldehyde at 223.5 nm is assigned to the ?T--?T*
allowed transition of the ethylenic double bond, which is
(IV) -Hz0 in conjugation with an aldehyde group (Fig. 2~).
Seventy percent GA solution, even if it is pure, con-
PI-
CHO CHO
+H,O
n
CHO CH(OH),
+H,O
G
U-QHC
n
CWW,
tains a considerable amount of glassy structures. When
this solution is diluted, although white turbidity appears
(1) (II) m 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 ex-
FHO fCH0 {PO tinction coefficient of the aldehyde peak is much lower
CH2-(CH2)2-CH C-(CHJ2-CH C-(CH2)2-CH0 than that of n-butyraldehyde, in the cases of both 70%
WI) solution (extrapolated to the zero time in Fig. 3A) and
0.4% solution (extrapolated to the infinite time in Fig.
FIG. 1. Possible molecular structures of GA in aqueous solutions 3A). This fact indicates that, although large portions of
and the reaction paths between them.
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.
3.5-4.0) were used as samples without purification. Ul- Since the hydration equilibrium constant for GA is not
traviolet absorption of GA was measured with a Beck- expected to be very different from those for aliphatic
man DU-70 spectrophotometer equipped with a thermo- monoaldehydes, the low abundance of the free aldehyde
stating circulator, in cuvettes of 10 or 2 mm in path group in GA indicates that the structures IV or V are
length. Light scattering was measured at 25°C with an predominant in aqueous GA solution (discussed in de-
Otsuka DLS-700s light scattering photometer at 633 tail later). Moreover, the spectral change in the dilution
nm, calibrated with benzene (9). The optical clarifica- process indicates the difference in (the abundances of)
tion was performed with Teflon filters. The specific re- the molecular structures of GA between 70% and dilute
fractive index increment (dnldc) was obtained with a solutions (described later).
Chromatix KMX-16 refractometer at the same wave- The shape of the absorption spectrum of 20% GA so-
length, calibrated with NaCl solution. The deoxygena- lution was almost the same as that of more dilute GA
tion of redistilled water, which was used to dilute the GA solution (data not shown) and underwent only a minor
solution, was performed by replacing the gaseous oxy-
gen in the water with gaseous nitrogen.
0.25 1"
RESULTS AND DISCUSSION
0.2
Figure 1 shows the possible molecular structures of
OJ
GA and includes all of the structures that have been 0
referred to in previous studies (l-5). We also propose z 0.15
here the most probable reaction paths deduced from the a
L
properties of aldehydes; there have been some discrep- =: 0.1
ancies also in the reaction paths that connect them. VI
s
is the average structure of the unsaturated polymerized
0.05
GA. (The number of methylene groups between the
neighboring ethylenic double bonds might be 1,2, or 3,
I
but the weighted average is exactly equal to 2.) VI might O 210 230 250 270 290 310
also contain ring structures at the end of its main chain.
The pendent aldehyde groups of VI would be scarcely Wave1 ength (nml
hydrated since the carbonyl form is stabilized by conju- FIG. 2. Ultraviolet absorption spectra of GA and its related com-
gation (10). pounds in H,O: (a) 16 mM n-butyraldehyde. (b) 5 mM crotonaldehyde.
Figure 2 shows the uv absorption spectra of some al- (c) 0.01 mM crotonaldehyde. (g) 70% commercial GA, preincubated at
2O”C, was diluted with deoxygenated water to 0.4% concentration (40
dehydes and GA in its dilution process. The wavelength mM) and measured at 2, 7, 12, 17, and 22 min after dilution with
of maximum absorption is shifted and the extinction increasing absorbance. The temperature was kept at 20°C after dilu-
coefficient is increased for the aldehyde group in cro- tion also.
96 KAWAHARA ET AL.

ments, we neglected the correction for isothermal com-

o-24o- pressibility 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
I 25’C. The same results were obtained for pH 7.0 and
$ 0.140'
6.0. Moreover, M.,, of GA in acidic solutions was also
z 0.200
shown to be ca. 100 (Fig. 6). M, did not change for more
s than a week despite the heat treatment. These facts indi-
0.195 cate that, contrary to earlier reports, dilute GA is almost
, B monomeric not only in acidic solutions but also in neu-
0.19ob , I tral and slightly basic solutions for an extended period
50 100 150 200 250 of time.
Ti me (mi n I When the results obtained from uv absorption and
FIG. 3. The absorbance variation of GA at 280 nm after dilution. light scattering are combined, it seems probable that
(A) The condition is the same as that for Fig. 2g, except that the most of the GA takes the structure of IV and the amount
absorbance is plotted against the time after dilution. (B) The condi- of V is negligible in dilute solution. On the other hand,
tion is the same as that for (A), except that ca. 20% commercial GA the gradual increase of absorption at 280 nm in the dilu-
was used as starting solution.
tion process of 70% GA indicates that 70% aqueous GA
contains a considerable amount of V structure, which
change in the dilution process (Fig. 3B). This indicates converts to monomer in the dilution process.
that the structure of GA in 20% solution is similar to Cross-linking reaction with proteins is usually carried
that in more dilute solution. out in less than 1% GA concentration. Under such con-
The comparison of the absorption spectra of GA with ditions, considering the effect of GA concentration on
those of crotonaldehyde (Figs. 2b and 2c) indicates that the equilibrium between the monomeric and the poly-
the unsaturated structure scarcely exists over a wide meric states, the concentration of polymeric GA is ex-
range of GA concentration, or in the transient state of pected to be even lower than that in the present study.
the dilution process. Although a small shoulder is ob- Since the conversion of V to monomers is rather slow
served at ca. 235 nm in the spectra of GA, and even if it (Fig. 3A), more attention should be paid to the time
is assumed to be due to the existence of the conjugated after dilution, when quantitative results are necessary
unsaturated structure, the content would be far below (for example, in the case of tissue fixation, where the
0.1% by weight, since crotonaldehyde (Fig. 2c) and simi- control of the osmotic pressure is important).
lar structures have strong absorption near this wave-
length. (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 hy- 1.4 -
drocarbon chain is attached to a-carbon in the case of &
VI (13)). It should be noted, however, that some com- z
mercial GA samples do contain impurities with signifi- 29 1.2 -
cant absorption at 235 nm (data not shown). e
The molecular weight of GA in aqueous solution was s
determined with light scattering. Figure 4 shows the - 1.0 -
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 I
(Kc/R,), (plotted as a,). This shows that the weight- -0.8 -0.4 0 0.4 0.8
average molecular weight (M,) of GA, which is equal to Sin* (e/21-1Oc
the reciprocal of (Kc/R,),, is ca. 100. Since i& is very
FIG. 4. The Zimm plot for GA in H,O. The concentrations are 1.78
sensitive to the existence of high-molecular-weight spe- (a), 3.62 (b), 5.25 (c), and 7.03% (d) (w/v), with a dnldc value of 0.175
cies, this clearly indicates that GA is almost monomeric ml - g-l. Each concentration was prepared by diluting 70% commer-
in 2-7% aqueous solution. In light scattering measure- cial GA with redistilled water several hours before measurements.
 ULTRAVIOLET INVESTIGATION OF GLUTARALDEHYDE STRUCTURE 97

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,, respec-
tively. 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), respec-
tively. (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
1.1I’ 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

1:
*0
in dilute GA solution, values of 7.5, 6.8, and 1.7% are
obtained for the concentrations of I, II, and III, respec-
tively, through the calculation mentioned above. How-
0.4 0.8 ever, if we consider the fact that the concentration of
si n2 (B/2) water is significantly lower in this case, the equilibria
FIG. 5. The molecular weight of GA in neutral or slightly basic between I, II, and III are expected to be considerably
aqueous solutions: 70% commercial GA was diluted to 3.4% (w/v) shifted to the left in Fig. 1, and the amounts are ex-
concentration with redistilled water and stored at room temperature pected to be between 7.5 and 11% (probably nearly
for several hours. The solution was then mixed with the same volume
ll%), less than 6.8% (probably considerably less than
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 6.8%), and less than 1.7% (probably nearly O%), respec-
min after (C) the pH adjustment. tively. 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 consider-
ably by the shifts of equilibria to the right in Fig. 1 in-
In aqueous solutions the possible interactions be-
tween the free or hydrated aldehyde groups in the same
GA molecule are expected to be mostly suppressed by
the interactions (hydrogen bond formation, electro-
static interaction) between each aldehyde group and
water molecules, except the formation of cyclic hemi-
acetal structure. Under such conditions, we may treat
120
the aldehyde groups of the GA molecule as independent
of each other when we consider the extinction coeffi-
cient and the equilibria among I, II, and III. With this
hypothesis the relative amount of each structure (in s 100 * ;
monomer units) in aqueous solutions can be estimated. “pi
Since c- of the free (not hydrated) aldehyde group of I
acetaldehyde, propionaldehyde, n-butyraldehyde, and i- 80 c
butyraldehyde were shown to have similar values with f
X, of ca. 280 nm in H,O (7), c280nm of the free aldehyde
group of GA is also expected to have a comparable I I I I
value. The extinction coefficient of the hydrated alde- 0 2 4 6 8 10
hyde is virtually zero at this wavelength (7). Thus, from
the absorbance A = 0.220 (obtained from the extrapola- Ti me (d 1
tion to the infinite time in Fig. 3A), ca. 16% of the alde- FIG. 6. The molecular weight of GA in acidic aqueous solutions.
hyde groups are estimated to be free in dilute solution at 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
20°C. Moreover, the equilibrium constant for hydration adjustment to 5.0 (O), 4.0 (A), and 3.0 (X), respectively. At the point
(K,,) does not depend strongly on the aliphatic chain indicated by the arrow, the temperature of the three sample solutions
length in monoaldehydes (7). Therefore, if we use Ki., of was elevated to 60°C for 1 h.
98 KAWAHARA ET AL.

duced by the significant increase of water activity in the CONCLUSION

dilution process, the amount of V is expected to be con- The structure of glutaraldehyde in aqueous solutions
siderably larger than 32.5% (l-0.1486/0.220 = 0.325). was investigated with uv absorption and light scatter-
The rest takes structure IV. It seems that, in 70% GA ing. Seventy percent glutaraldehyde solution contains a
solution, not only the high concentration of GA but also large quantity of polymeric species with cyclic hemiace-
the low concentration of water play an important role in tal structure. On dilution the polymerized glutaralde-
the formation of V and in other shifts of equilibria from hyde slowly converted to monomers. In dilute solution,
the state of dilute solutions. glutaraldehyde is almost monomeric at pH 3-8, the ma-
Korn et al., who also studied the 70% GA solution, jor portion taking the cyclic hemiacetal structure. The
suggested a value of 15% for I and 85% for (IV + V) at structure of GA in 20% solution is similar to that in
25”C, on the basis of their H-NMR experiments (4). more dilute solution. a&Unsaturated structure does
(They also suggested the possibility of a small amount not exist in aqueous solution regardless of the concen-
of II and III). Considering the effect of temperature on tration of glutaraldehyde.
the equilibria between molecular structures of GA (4),
their results suggest that the expected amount of I is ACKNOWLEDGMENT
less than 15% at 20°C. Since the sample used by Korn et
The authors thank Drs. Hideo Orita, Masao Shimizu, Akiko Ta-
al. is unlikely to have had exactly the same composition katsu, Masaaki Sugiura, and Arnfinn Andersen for their helpful dis-
as that used here (the actual concentration of commer- cussion. This work was supported in part by special coordination
cial 70% GA solution is not necessarily 70%, and this funds for promoting science and technology from the Science and
deviation of the concentration can cause a slight varia- Technology Agency, Japan.
tion of the composition), the reported values are in good
agreement with the present study. REFERENCES
For those using GA as a cross-linking reagent for pro- 1. Richards, F. M., and Knowles, J. R. (1968) J. Mol. Biol. 37,231-
teins, the following comments might be useful. It is well 233.
known that GA cross-links proteins very effectively. As 2. Hardy, P. M., Nicholls, A. C., and Rydon, H. N. (1969) &em.
a matter of fact, this monomeric GA (diluted from 70% Commun., 565-566.
commercial GA and stored at room temperature for sev- 3. Whipple, E. B., and Ruta, M. (1974) J. Org. Chem. 39,1666-1668.
eral hours) cross-links myosin molecules to form a gel in 4. Korn, A. H., Feairheller, S. H., and Filachione, E. M. (1972) J.
Mol. Biol. 65,525-529.
GA concentrations as low as 0.01% (data not shown). It
5. Monsan, P., Puzo, G., and Mazarguil, H. (1975) Biochimie 57,
might seem rather difficult to explain this effectiveness
1281-1292.
of GA in cross-linking reactions, if principal species of
6. Peters, K., and Richards, F. M. (1977) Ann. Reu. Biochem. 46,
GA in aqueous solution are monomers. Our results, how- 523-551.
ever, do not necessarily indicate that GA cross-links 7. Gruen, L. C., and McTigue, P. T. (1963) J. Chem. Sot., 5217-5223.
proteins in the monomeric form. Rather, there is a possi- 8. Silverstein, R. M., Bassler, G. C., and Morrill, T. C. (1981) Spec-
bility that polymeric structures like VI are involved in trometric Identification of Organic Compounds, 4th ed., pp. 249-
the actual cross-linking reactions, since in the reaction 303, Wiley, New York.
of GA with a trace amount of n-amylamine, which is 9. Pike, E. R., Pomeroy, W. R. M., and Vaughan, J. M. (1975) J.
&em. Phys. 62,3188-3192.
considered an analogue of the side chain of Lys residue,
10. Hooper, D. L. (1967) J. Chem. Sot. B, 169-170.
a significant absorption peak that closely resembles
11. Greenzaid, P., Luz, Z., and Samuel, D. (1967) J. Am. Chem. Sot.
that expected for the x--x* transition of the ethylenic
89, 749-756.
double bond in the structure VI is produced (data not
12. Bell, R. P. (1966) Adu. Phys. Org. Chem. 4,1-27.
shown). In other words, it could be that monomeric GA
13. Fieser, L. M., and Fieser, M. (1949) Natural Products Related to
is converted to polymeric forms by the action of amino Phenanthrene, p. 184 ff., Reinhold, New York.
group, and this newly produced polymeric GA plays a 14. Zimm, B. H. (1948) J. Chem. Phys. 16, 1099-1116.
major role in the cross-linking reaction of proteins. This 15. Kamata, T., Nakahara, H., and Hattori, S. (1977) Bull. Chem.
cross-linking mechanism of GA is the next important Sot. Jpn. 60,2558-2563.
problem to be elucidated and now is under investigation 16. Kamata, T., and Nakabara, H. (1973) J. Colloid Znterface Sci. 43,
in our laboratory. 89-96.