trends in analytical chemistry, vol. 13, no.
70, 1994
1.J.B. Meuwsen is at the Pharma Bio-Research
Labora tories B. V., Assen, The Netherlands.
M.L. Salm is at the Chief lnspectorate of Health
The chemistry of enzyme and protein
immobilization with glutaraldehyde
David R. Walt *, Venetka I. Agayn
Medford, MA, USA
Immobilization of proteins to solid matrices
has been performed for the last thirty years
and has provided numerous examples of
successful preparations with use in enzyme
reactors, sensor preparation and immunodi-
agnostics. Among the arsenal of coupling
reagents and procedures, glutaraldehyde
plays a critical role due to its reliability and
ease of use. It displays a complex chemistry
that is transparent to most practitioners of
immobilization. In this article we detail the
structure and reactivity of glutaraldehyde
protein immobilization.
1. Introduction
Of the many commercially available cross-link-
ing agents, glutaraldehyde (pentandial) has
undoubtedly found the widest applications. It is
used to immobilize proteins to solid supports for
use in enzyme reactors, affinity chromatography,
in immunochemical research, and as a cross-link-
ing agent for biosensors. Glutaraldehyde (GA) is
a generally dependable material that invariably
gives high yields of immobilized protein on the
support. Two principal procedures exist for accom-
plishing immobilization with GA including:
0 activation of the support followed by treat-
ment with the protein (e.g., polyacrylamide
and controlled-pore glass) ;
0 protein cross-linking in which GA acts to
cross-link proteins thereby czausing them to
gel (e.g., membranes for enzyme electrodes
[ 1 ] and fiber optic sensors [ 2,3] ).
* Corresponding author.
0 1994 Elsevier Science B.V. All rights reserved
425
Protection, State Inspectorate of Public Health
(Ministry of Welfare, Health and Cultural Affairs),
Rijswijk, The Netherlands.
Despite the success of this reagent, the proce-
dures employed have been developed largely
through empirical methods rather than with a full
understanding of the nature and chemistry of the
reagent. It is the intention of this article to clarify
some of the chemistry of glutaraldehyde and to
propose mechanisms through which immobiliza-
tion occurs. Our purposes here are to review the
literature on the structure of GA and to speculate
on its nature so as to provide the protein immobi-
lization practitioner with guidelines regarding the
chemistry of this reagent.
2. History of GA uses
Glutaraldehyde has been used as a fixative for
decades [4]. Its ability to ‘fix’ tissue has to do with
its bifunctional nature resulting in its cross-linking
capability. Proteins within tissue become cross-
linked leading to a tissue ‘polymer’ that cannot be
dissolved or dissected into its components except
by gross mechanical means. Initial studies on the
chemistry of cross-linking with GA were prompted
by its use for preparing stable crystals for X-ray
diffraction studies [ 51 or fixing tissue samples for
microscopic investigation [ 61. It was established
that treatment of the fragile protein crystals with
GA renders them mechanically stable and insoluble
in 1 A4 NaCl, however, the treated samples still
retained approximately 30-70% of their original
enzyme activity [ 71. Such crystals develop a yel-
low color, however, treatment with NaBH, leads
to the disappearance of the color and a slight
increase in the enzyme activity [ 51.
GA has also found multiple uses in immuno-
chemistry:
0 to prepare adjuvants by cross-linking proteins
in order to render them more immunogenic
[81,
0165-9936/94/$07.00
426
0 to cross-link antibodies (Ab) or antigens
(Ag) absorbed on solid supports to enhance
their stability [9],
0 to fix cells on microtiter plates in the prepa-
ration of screening assays [ IO],
0 to conjugate Ab or Ag to enzymes for ELISA
assays [ Ill,
0 to prepare affinity columns in which Ags or
Abs are coupled to GA activated beads to
obtain highly specific immunoadsorbents for
isolating Ags or Abs in high yields [ 121.
GA also has been used extensively in enzyme
and viable and non-viable cell immobilization
[ 13,141. Among its many industrial applications
are the immobilization of lactase on carbon by
adsorption followed by GA cross-linking and the
use of GA immobilized penicillin acylase for the
production of 6-aminopenicillanic acid - a start-
ing material in the production of a number of p-
lactam antibiotics. Many different supports can be
used [ 151 in conjunction with GA including nylon,
fumed silica, controlled-pore glass, cross-linked
proteins such as gelatin and bovine serum albumin,
and polymers with pendant amino-groups. The
recovery of enzyme activity is different in each
preparation, but as a rule the enzymes are found to
be more temperature stable, more resistant to dena-
turation and more stable to prolonged use.
GA is used routinely in research for the prepa-
ration of biosensors in which enzymes or cells are
attached to transducers such as optical fibers or
electrodes [ l-31. Enzyme electrodes and therm-
istor probes have been prepared by immobilizing
alcohol oxidase on the electrode [ 161; sensors also
have been prepared by immobilizing alkaline phos-
phatase [3] or penicillinase [ 21 on glass optical
fibers.
A different application of GA’s ‘fixing’ capabil-
ities is its use in preparing microcapsules (or
microspheres) from proteins such as gelatin or
albumin for drug delivery devices. In this case GA
cross-links the protein spheres to ‘harden’ them
[171.
All these diverse applications underscore the
importance of understanding the chemistry of GA
to achieve the best results in each particular
instance. A substantial literature exists regarding
the structure and use of GA [ 7,18-201. Active
research continues to provide new examples and
applications of this reagent.
In addition to the published literature, a great
deal of anecdotal information exists concerning the
use of GA. When GA was first used for immobili-
trends in analyticalchemistry, vol. 13, no. 70, 1994
zation, many thought that its cross-linking ability
was due to Schiff base formation between lysine
residues on proteins and the aldehyde groups on
GA. This notion persists even today [ 2 I]. These
Schiff bases, however, are known to be highly
labile and would be expected to be reversible under
most aqueous conditions [22]. Because of this
lability, the common protocol was to reduce the
incipient Schiff base with NaBH4 or NaB (CN) H3
to an amine. Although this procedure is practised
routinely even today, there is little evidence that
such reduction enhances the stability of the immo-
bilized protein. In fact, it is well known that pro-
teins immobilized with GA are sufficiently stable
even in the presence of exogenous amine to confirm
that the Schiff base mechanism cannot be operative
[ 71. Furthermore, the use of reducing agents might
be inappropriate in cases where disulfide bridges
in the protein are responsible for maintaining struc-
tural integrity and reactivity.
Another mystery regarding GA is its enhanced
effectiveness upon standing. Many experienced
immobilization chemists ignore the label on the GA
container that recommends it be stored at 0°C and
leave it out on the lab bench before ever using it
for immobilization. This practice is believed to
allow GA to react intermolecularly to form poly-
meric structures which provide a reagent with
enhanced cross-linking and immobilization capa-
bility. Although such practice leads to an efficient
protein immobilization, freshly distilled GA is also
highly effective.
Finally, GA obviously possesses unique char-
acteristics that render it a far more effective cross-
linking reagent than other dialdehydes, such as
glyoxal, malonaldehyde, succinaldehyde, etc.
[23]. We hope to clarify this unique chemistry in
the following section.
3. Structures of glutaraldehyde
The effectiveness of GA immobilization and the
controversies surrounding its chemical behavior
can be rationalized with a multiplicity of structures
that depend on the solution conditions. Different
studies [ 19,24-261 have shown that commercial
aqueous solutions of GA (25 or 70%) represent
multicomponent mixtures. The pH of these aque-
ous solutions is 3.1 which can be explained if some
of the aldehyde groups are partially oxidized to
carboxylic acids. NMR analysis identified the fol-
lowing GA structures in water: free GA (I), linear
trends in analytical chemistry, vol. 13, no. 10, 1994 427

gested that dilute solutions might improve the stor-

I\
OH OH
I II III
HO~"~oH
x
Fig. 1. Structures of GA in water solution.
mono- (II) and dihydrates (III), a cyclic hemi-
acetal (IV) and oligomers (V) (Fig. 1). Upon
raising the temperature, the amount of free GA
increases greatly at the expense of the cyclic
hydrates [ 24,251. In more concentrated solutions
(higher than 25%)) or under acid conditions, cyclic
hemihydrate multimers V can be formed. Upon
dilution with distilled water, however, these poly-
meric materials revert to a monomeric form of GA
as would be expected from the reversibility of the
hydration reaction. Therefore, it has been sug-
monomeric
age of monomeric GA [ 61.
Obviously, each structure would participate dif-
ferently in cross-linking reactions and result in dif-
ferent products. One could propose the products
shown in Fig. 2 upon reaction of these different
compounds with protein. Aldehydes would be
expected to form Schiff bases upon nucleophilic
attack by the lysine residues in the protein as shown
in Eq. ( 1) . Schiff bases are unstable under acidic
conditions and they break down to regenerate the
aldehyde and amine. With this in mind, many pro-
cedures have recommended the use of a reducing
agent such as NaBH, to convert the Schiff base to
a stable secondary amine. Some researchers have
found that preparations treated with reducing
agents exhibit higher activity than the untreated
ones. However, it has been established that GA
treated proteins do not regenerate lysine (6 MHCl,
110°C 24 h) [ 181. Hence, Schiff base formation
is not the likely mechanism of this reaction. A sub-
sequent reduction step would not be necessary to
stabilize either products IVa or Va of reactions (2)
or (3). Thus it is likely that under acidic conditions
monomeric and multimeric GA react via reactions
(2) and (3).
Basic conditions cause GA to undergo rapidly
intermolecular aldol condensations and result in
a,P-unsaturated oligomeric aldehydes of type VI
shown in Fig. 3. Their formation can be monitored
by an increase in absorbance at 235 nm, where pure
GA does not absorb. Structures VI were proposed
initially as the major constituent of pure GA solu-
tions [ 271, but the contemporary view attributes
their formation to a reaction of GA under basic

n
(1)
conditions. In these products, the internal aldehyde
CHO CHO
groups exist in conjugation with the C-C double
I
bonds and Schiff bases formed at these positions
(such as Via) would be greatly stabilized by res-
(2) onance thus explaining their acid stability. Upon

HO
CL o OH
reduction with NaBH, a stable secondary amine is
formed. Under excess of amine a second reaction
is possible, i.e., a Michael addition to the double
IV bond. This reaction would result in disruption of
the resonance stabilization and render the adduct
muhmeric
VIc labile to acid hydrolysis. Based on these reac-
(3)
tivity principles and from IR spectral evidence,
Monsan et al. [28] argue that the product of the
reaction of an Q-unsaturated aldehyde polymer
with amino groups in proteins is not a Michael
V ?
V&3 product but the imine stabilized conjugate Via.
Fig. 2. Reactions of GA with proteins under acidic or Others accept the original suggestion of Richards
neutral conditions. and Knowles that the polymeric unsaturated alde-
428 trends in analytical chemistry, vol. 13, no. 10, 1994

CHO CHO

CHO

_ x

VI

(1) NHzeEz

k-N
“C CHO - CHO CHO

CHO + OHC CHO

- x EZ’N H -x

Via
VIb

(2) NHy-“-Ez

Er %N

-“C -CHO

OHC CHO

EZ’# _ x

WC

Fig. 3. Reactions of polymeric GA with proteins under basic conditions.

hyde reacts in a Michael fashion to form a product
VIb which is stable to acid hydrolysis.
To complicate the matter further, another prod-
uct of the reaction of GA with proteins was sug-
gested to be quaternary pyridinium compound
VIII. Direct evidence for the product formed
between GA and lysine residues in proteins was
obtained in the work of Hardy et al. [29] who
isolated and identified a quaternary pyridinium
compound (Fig. 4). They suggested that this prod-
uct results from a reaction of the a&unsaturated
multimeric aldehyde with proteins to form a simple
Schiff base VII which can undergo cyclization,
dehydration and internal redox reactions.
It is interesting to speculate whether proteins can
catalyze the aldol condensation/polymerization
GA. Proteins contain both acid residues, such as
coupling GA with enzyme the maximum binding
was achieved at pH 7.7 for 2 h at 4°C. Activation
of the gel at a pH higher than 7.0 leads to extensive
aggregation of the beads, presumably due to
increased GA polymerization. These results are in
accord with the suggested structures for GA and
their behavior in reaction with proteins. Lower pH
is more appropriate for activation of the gel since
GA is more stable under these conditions (i.e. less
prone to polymerization), while higher pH is
required for efficient nucleophilic attack by the pro-
tein’s lysine residues.
NH-CH-CO . NH-CH-CO
of
I I
(CHzh
I Hz0 KHz)4

aspartic and glutamic acids, as well as base resi-

N\
dues, such as histidine and lysine. These residues :~,II,‘,:r~za,,o” ) (J--o

can catalyze aldol condensation via Knoevenagel o/lJ=ol

type condensations. Thus excess monomeric GA (CHzh

(CHd,
may polymerize upon exposure to the very protein
it cross-links! ._.._NH-CH-CO _.__ ,,... NH-CH-CO _.__

A study of the effect of pH on the immobilization

efficiency of acid phosphatase [ 301 showed that \I1 VIII

the optimal pH for activating polyacrylamide beads Fig. 4. Cross-linking with GA through quaternary pyr-
with GA is 6.9 at 4°C for up to 40 h, while for idinium compound.
trends in analyticalchemistry vol. 13, no. 10, 1994 429

IX

Fig. 5. Structure of polymeric GA product of incomplete condensation undergoing a Cannizzaro reaction.

More recent studies suggest polyGA is a more
efficient cross-linking reagent than monomeric
GA. PolyGA was used to immobilize antibodies
on soluble substrates [ 3 11. PolyGA was prepared
by allowing GA solutions to react at pH 7.0-13.5
resulting in either soluble or insoluble reactive pol-
ymers with a variable number of hydrated and non-
hydrated aldehyde groups that may be conjugated
with ethylenic linkages. Due to incomplete dehy-
dration of the aldol products, secondary hydroxyl
groups may be present. Cannizzaro reactions also
CHO
OHC
VI
/
OHC
/
occur with free aldehyde groups producing both
primary hydroxyls and carboxylic acid groups in
these polymers (Fig. 5).
During the aldol condensation of GA solutions,
oligomers are also formed with improved cross-
linking characteristics [ 321. Alkali treated solu-
tions of GA are reported to result in higher activity
of immobilized alcohol dehydrogenase and gluta-
mate dehydrogenase than untreated GA. The oligo-
mers are considered to be of the structure VI shown
in Fig. 3. A recent study [33] identified dimers X
and XI as major components in the alkaline acti-
vated GA as shown in Fig. 6. Reaction of X and
XI with proteins may involve a Michael addition
CHO to the double bonds to give Xa and XIa. If these
reactions occur, no reduction with NaBH, or
NaB( CN) H, is necessary to stabilize the adducts.

CHO
m OH ===== oHcaOH 4. Conclusions
X XI

GA is the reagent of choice in many applications

ErmNH> EzmNH2
for fast and dependable protein immobilization. It
I I is inexpensive, readily available and easy to use.
EZ EZ
‘NH ‘NH GA exists in different forms depending on the solu-
OHC OHC
tion conditions. Under acidic and neutral solutions
CHO -
GA exists as a monomer in either its free aldehyde
tl? ti OH
0
OH
form, hydrate or hemiacetal. At higher concentra-
Xa XL3 tions it polymerizes to oligomeric hemiacetals. All
EzmNHn of these forms can react with proteins in different
/
ways and lead to immobilization. Under basic con-
EZ
‘NH ditions GA undergoes aldol condensations to form
OHC a&unsaturated multimeric aldehydes. These
NrEZ
products react with proteins either via stabilized
to.
o H Schiff base formation or by a Michael adduct for-
Xlb mation. Polymeric GA in either linear or cyclized
Fig. 6. Reactions of dimeric cyclic GA with proteins forms shows improved immobilization capabili-
under basic conditions. ties. The protocol for using GA should be specified
430
for each particular protein and application. Despite
protocol differences, there are several general use-
ful guidelines for the practitioner:
0 GA should be stored in dilute solutions at low
temperature and at pH 3-4 to prevent poly-
merization.
0 Activating the support for immobilization,
should be conducted at a lower pH, (e.g. pH
6.8) while the protein coupling should be car-
ried out at an elevated pH to promote nucleo-
philic attack and improve immobilization.
0 The use of a reducing agent is usually not
required and might prove disadvantageous for
the particular application. The necessity for
reduction should be confirmed before it is
incorporated into a particular protocol.
0 The use of polymeric GA may be advanta-
geous. Caution should be used however, since
the polymeric form will have large dimen-
sions and lower diffusion rates which may be
a disadvantage in certain applications (e.g.
when fixing tissue samples or activating bulk
polymer matrices).
The complexity of GA should not discourage its
use for immobilizing proteins; in fact, all known
forms exhibit the ability to react and cross-link
proteins. By judicious choice of storage and reac-
tion conditions, GA can be used successfully for
many protein immobilization needs.
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David R. Walt is a professor and Chairman of the
Department of Chemistry at Tufts University,
Medford, MA 02155, USA. His scientific interests
include optical sensors, enzyme-catalyzed
synthesis and bioma terials.
Venetka I. Agayn is a graduate student at Tufts
University and her interests are in bioprocess
control, optical sensors and immunoanalysis.