Chemical crosslinking and the stabilization of proteins and enzymes Shan S. Wong* and Lee-Jun C. Wongt * Department o f Pathology and Laboratory Medicine, University o f Texas Health Science Center at Houston, Houston, Texas, and t Institute for Molecular Genetics, Baylor College o f Medicine, One Baylor Plaza, Houston, Texas
The technique of chemical crosslinking has been used to enhance the stability of proteins and enzymes. In this procedure, the molecule is braced with chemical crosslinks either intramolecularly or intermolecularly to another species to reinforce its active structure. Various chemicals have been used for this purpose. The bifunctional reagents are the most prominent. These compounds are derived from groupspecific reagents and may be classified into homobifunctional, heterobifunctional, and zero-length crosslinkers. Different physical and chemical characteristics have been incorporated into these chemicals. Their versatility holds great potential in preparing chemically, thermally, and mechanically stable proteins and enzymes for industrial applications.
Keywords: Protein stability; chemical crosslinking, crosslinking reagents; enzyme activity; bifunctional reagents
Introduction The stability of proteins and enzymes is a major concern in their industrial applications. Biocatalysts with high thermostability will have a prolonged viability, t A lengthened operational stability will reduce the need for frequent enzyme replacement and thus decrease the cost of enzyme preparations, which can be rather expensive. 2 These thermostable enzymes will enable the acceleration of chemical reaction rates at a higher temperature. According to Van't Hoff's rule, an increase of temperature by 10°C will raise the rate of chemical reactions by two- to threefold. In cases where several reaction steps are required, a thermostable biocatalyst will enable the process to continue at elevated temperatures without lowering the heat. 3 Reactions occurring at higher temperatures will also shift the thermodynamic equilibrium. For endothermic processes, product formation will be favored at elevated temperatures, as stated by Le Chatelie's principle. Reactions at high temperatures also provide a simple termination procedure by cooling. Not only high thermostability is desirable, but also stability in organic solvents, in extreme pH values and pressure, and towards mechani-
Address reprint requests to Dr. S. S. Wong at the Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030 Accepted for publication 14 June 1992
866
cal disturbances is required for organic synthesis, chemical analysis, isolation, and purification of chemicals, in therapeutics and diagnostics, and in the study of protein structures and functions. These and other advantages have prompted a persistent search for methods to prepare stable proteins and enzymes.
Chemical basis of protein stability Recent interests in structure-stability relationships in proteins have led to the calculations of free energies contributed by various structural determinants. Numerous articles and reviews have been published. 4-H A brief summation is included here for the understanding of the approaches undertaken to prepare stable proteins and enzymes. Hydrophobicity has always been considered one of the major factors of protein integrity. 12.13Hydrophobic side chains are usually buried inside the protein molecules to induce a tight packing. For proteins that associate with other components, hydrophobic clusters are found localized on the surface of the protein molecules to induce solitary contacts with other proteins, with lipids, or with other hydrophobic entities. 14 This shielding of nonpolar groups from interaction with water in the medium reduces the unfavorable entropy of the system and thus increases protein stability.15-~7 In addition to hydrophobic forces, hydrogen bonding, salt bridges, dipole-dipole, and other electrostatic interactions also contribute to protein stability. 18 Hydrogen bonds play an important role in the maintenance of
Enzyme Microb. Technol., 1992, vol. 14, November
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Stability and crosslinking: S. S. Wong and L.-J. C. Wong secondary structures of proteins, i.e., o~-helixes, /3sheets, etc. They also provide an interaction of polar groups of protein and water at the protein-water interface to form a hydration sphere of the molecule. 14While the energy contribution of dipole-dipole interactions is small, the large number of such interactions in proteins makes it significant.19 On the other hand, the number of salt bridges is small, but their energy contribution is large. 2°,21 It is the sum of these various forces, together with the binding of metal ions, substrates, cofactors, and other low-molecular-weight ligands, that maintains the structural integrity of a functional protein or enzyme. 22 Although the molecular mechanism of protein inactivation may involve several conformational changes leading to the unfolding of the molecule,23'24 the overall physical process may be represented simply by two steps: N ~ I---~ D where N, I, and D are native, intermediate, and denatured forms of the protein, respectively. 23-26 The first step involves reversible conformational changes, followed by a second step of irreversible unfolding by which an enzyme loses its activity. The approach to stabilizing proteins and enzymes is to prevent irreversible unfolding from occurring. 27-31This can be achieved by several means.
Methods of stabilization Many practical methods have evolved to preserve the integrity and activity of the native proteins. Based on thermodynamic reasoning, proteins were invariably kept at low temperatures to prolong their longevity. Although many enzymes and proteins can be stored in this manner for an extended period of time, low temperatures are not preferable in many industrial operations. Furthermore, some proteins are cold-sensitive and may be more stable at ambient temperatures. In an attempt to mimic the microenvironment of the proteins in the cell, various substances have been added to interact with isolated proteins. Glycerol, ethylene glycol, and detergents have been used to preserve enzyme activities after isolation, particularly enabling the proteins to be kept at very low temperatures without being frozen. 32 Other substances, such as carbohydrates (e.g., sucrose) and proteins (e.g., albumin), have also been used to stabilize proteins of interest. While these methods have preserved the biological activities to various extents, the procedure introduces foreign substances, which may not be desirable in many applications. A technique to increase the contributions of various forces to the free energy of stabilization is protein engineering, involving site-specific mutagenesis and cloning of thermostable molecules whose structures mimic that of proteins isolated from thermophilic organisms.33 This method seems to be appealing, but tremendous difficulties associated with protein characterization, including three-dimensional structural analysis, will have
to be resolved before site-specific mutagenesis can be defined. Another approach to stabilizing proteins and enzymes is to strengthen the compact structure of the molecule so that denaturation will not occur. This may be accomplished by chemical crosslinking. It has been demonstrated that intramolecular disulfide bonds contribute significant stabilization energy. Doig and Williams 5'34'35 have argued that disulfide bonds destabilize folded structures entropically, but stabilize them enthalpically to a greater extent, resulting in an overall stabilization of the molecule. Balaji et al. 36 have used x-ray crystal structure data to define geometric parameters in serine proteases and subtilisin to introduce disulfide bridges which lead to thermal stability. Like disulfide bonds, chemical crosslinking reagents have been used to brace proteins intramolecularly and to conjugate them intermolecularly to other molecules, including solid supports. This technique has been demonstrated to greatly enhance the stability of proteins.
Stability effected by chemical crosslinking If the molecular basis of inactivation is a reflection of the disruption of the secondary and tertiary structure of a protein, the active conformation may be reinforced with chemical crosslinking reagents. As depicted schematically in Figure 1, chemical crosslinks will reticulate the molecule to diminish the polypeptide entropy, which will decrease the rate of denaturation. With multiple attachments, the molecule is held in its original active structure. The braces can be achieved by both intra- and intermolecular crosslinking. Many studies have shown that intramolecular crosslinking is effective in protein stabilization. Shaked and W o l f e 37 have
CTV Figure 1 Stability by intra- and intermolecular crosslinking. (A) Intramolecular crosslinks reticulate the molecule to reinforce the structure. Multipoint intermolecular crosslinking (B) to a solid support and (C) to a soluble entity has a similar effect
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Review used dimethyl suberimidate and dimethyl adipimidate to crosslink catalase. The process removed 44 of the reactive amino groups and showed impressive stability towards glucosone and temperature. Olsen e t a / . 38-4° have crosslinked hemoglobin with bis(3,5-dibromosalicyl) fumarate. A single crosslink between Lys82/31 and Lys82/32 increased the denaturation transition temperature of methemoglobin A from 40.7 to 57. I°C. For cyanomethemoglobin the transition increased from 55.5 to 67.5°C. With deoxyhemoglobin, a crosslink between Lys99al and Lys99a2 was obtained. The a crosslinked hemoglobin has the same stability as the/3 crosslink, with a gain in transition temperature of 17°C. Since the amino acid residues modifiable by crosslinking reagents are distributed randomly in a protein, their positions in the three-dimensional structure differ in space and microenvironment. Different crosslinking reagents may vary significantly in their ability to confer stability. Tatsumoto et al. 41 have used a series of bifunctional chemical modification reagents of different chain lengths to crosslink amyloglucosidase. Several of the diimidoesters were shown to more than double the half-life of this enzyme at 65°C, but dimethylglutarimidate provided the longest half-life. Torchilin et al. 42 have reported that the degree of increased thermostability of a-chymotrypsin depends on the length of carbon chain in the intramolecular crosslinks. The highest stabilizing effect is produced by carbodiimide-activated enzyme with 1,4-tetramethylenediamine. Similarly, intramolecularly crosslinked glyceraldehyde-3phosphate dehydrogenase is more stable at 60°C than native enzyme, and the degree of stabilization is dependent on the chain length of the bifunctional reagent u s e d . 43 Maximal thermostabilization was found when succinic acid was used in the carbodiimide-activated enzyme. Multipoint fixation of enzymes to solid supports or soluble polymers has been shown to increase the resistance of enzymes and proteins to high temperatures and denaturants such as u r e a . 28'33'44'45 Crosslinking of superoxide-generating respiratory burst oxidase in neutrophil plasma membranes with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, has significantly enhanced enzyme stability towards heat, high salt, and detergent. 46 The crosslinked enzyme has an increased half-life from 2 to over 20 min at 37°C, and is completely active in 0.1 M KCI and 0.4% Triton X-100, which have inactivating effects on the oxidase. Intermolecular crosslinking of enzymes of interest to other soluble proteins is also beneficial. Such an indication was first demonstrated by ribonuclease when it was crosslinked with dimethyl suberimidate to itself to form a dimer or to polyspermine to yield a heteroconjugate. 47'48 The polysperimine-ribonuclease conjugate was 115 times more active towards poly(A) • poly(U), and 176 times more active towards poly(I). poly(C) than the parent enzyme. 48 Further studies revealed that when horseradish peroxidase is crosslinked to immunoglobulin G or Jacalin, the energy of inactivation of the enzyme increased from 35 to 51 and 43 Kcal
868
mo1-1, respectively. 49 Recently, Wang et al. 45 have synthesized a series of aminoglucose-based carbohydrate polymers which have been demonstrated to stabilize proteases in organic solvents and elevated temperatures. Immobilization of molecules to solid supports has long been recognized to confer stability. 27-29A severalhundred- to a thousandfold decrease in both the reversible and irreversible thermoinactivation of enzymes has been observed. ~.30,3~.50Most strikingly, highly heat-stable thermophilic asparaginase and caldolysin can be additionally stabilized 10 times by immobilization. 5~'52 Stability towards denaturants such as urea has also been noted. 53 Demers and W o n g 54 have demonstrated that immobilization can also increase the mechanical stability, as shown for galactosyltransferase. Thus, there are ample examples to illustrate that chemical crosslinking, either inter- or intramolecular, provides a means of preparing stable enzymes and proteins.
General characteristics of crosslinking reagents Chemical crosslinking of proteins and enzymes can be most easily achieved with bifunctional compounds in comparison to monofunctional reagents. 5°,55-6° The bifunctional reagents are essentially chemical modification agents and may be classified into zero-length, homobifunctional, and heterobifunctional crosslinkers. These chemicals react with nucleophilic side chains of amino acids, such as the sulfhydryl group of cysteine, the amino groups of lysine and the N-terminal amino acids, the carboxyl groups of aspartic and glutamic acids and the C-terminal amino acids, the imidazolyl group of histidine, and the thioether group of methionine. The specificity of these chemicals for a specific amino acid side chain depends on the relative reactivity of the nucleophile. Since the nucleophilicity is a function of the electronic structure, the P K a , and the microenvironment, the reactivity of an amino acid side chain is generally not specific, and several side chains may react with the same bifunctional reagent. 6° In general, however, the thiolate ion is the most nucleophilic. 61 Thus the sulfhydryl groups of proteins will react with most of the crosslinkers at alkaline pH. For example, a-haloacetyl compounds and N-maleimido derivatives are generally considered as sulfhydryl-selective. A different set of reagents that are thiol-specific are disulfide and mercurial compounds. Disulfide compounds react through a disulfide interchange reaction, as shown in Figure 2. 62
In proteins where the free sulfhydryl moiety is not available, the amino group becomes the major target of reaction. Also, for proteins with high surface amino group content, the competition for the reagent may become favorable. In addition, thioacyl esters, as a result of acylation, are susceptible to hydrolysis. Thus, acylating agents are in general considered amino groupdirecting. Of the crosslinking reagents, the zero-length crosslinkers induce direct joining of two chemical compo-
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and crosslinking:
@s-cl
_!-.N-
S-SONO,
NOz
@s-s--R
N2S-ScN
ai,-s
-s
gNo2
COOH
coon A
0
C
Figure 2 Disulfide interchange reaction. The free thiolate ion displaces another thiol from a disulfide compound. Examples of disulfide reagents are: (A) 5,5’-dithiobis-(2nitrobenzoic acid); (5) 4,4’-dithiopyridine, and (C) methyl-3-nitro-2-pyridyl disulfide
nents without the introduction of any extrinsic material. This is in contrast to homo- and heterobifunctional reagents, where a spacer is incorporated between the two crosslinked groups. Examples of zero-length crosslinkers are carbodiimides, isoxazolium derivatives, chloroformates, and carbonyldiimidazole,63-66
S. S. Wong and L.-J. C. Wong
which couple carboxyl and amino groups, and cupric di( 1, IO-phenanthroline) and 2,2’-dipyridyldisulfide, which form disulfide bonds. These reagents react by activating one of the moieties to form an active intermediate, which then reacts with a second component, as shown in Figure 3. The other classes of crosslinkers contain two reactive functionalities which will react with amino acid side chains, thus bringing the two components together. The reactive groups are located at the two ends of the molecule, connected by a backbone which may be designed to contain specific characteristics. Because the most reactive amino acid side chains susceptible of modification are nucleophiles, acylating and alkylating agents are incorporated into these head groups.S*67 When a crosslinker contains two identical reactive groups, it is referred to as a homobifunctional reagent. Examples of diacylating agents include bisimidoesters, bis-succinimidyl derivatives, di-isocyanates, di-isothiocyanates, di-sulfonyl halides, bis-nitrophenyl esters, dialdehydes, and diacylazides.N A few common representative compounds are shown in
_oP,
=C=N--_R’
Ii c-o -c-“-“’
\,
Figure 3 Examples and mechanism carbonyl diimidazole
of action of zero-length
crosslinkers:
(A) ethyl chloroformate,
Enzyme Microb. Technol.,
(6) a carbodiimide,
and (C) l,l’-
1992, vol. 14, November
889
Review O
O l'
N
O n --O
O
A
O l. ' L t --N O
+ + . I~H,CI ~H ,Cl CH=--O --C --CH:l--C-- O~CH=
B
O=C=N----~.=C=O
S=C=N---O--.=C=S
C
D
j,o.c,
e.'-i ~ ' !
"~
0
Figure 4 Examples of diacylating homobifunctional reagents: (A) disuccinimidyl suberate, (B) dimethyl malonimidate, (C) 1,4-dicyanatobenzene, and (D) p-phenylene-diisothiocyanate. The mechanism for crosslinking is illustrated by phenol-2,4-disulfonyl chloride
O
O
~N--CHI~N~
ICH3i~CH=
O
O
I ,O3H
A
B O
c._c,.c.,_s_c.,c.,-c, '43-!-C~, 1 O
NO=
C
NO2
D
Qs-~+ ,~_~ . . _ i _ o ~ -
0
@,-c.,4-o.,-,@
Figure 5 Examples of dialkylating homobifunctional reagents: (A) N,N'-methylenebismaleimide, (B) e,~'-diiodo-p-xylene sulfonic acid, (C) di(2-chloroethyl)sulfone, and (D) bis(3-nitro-4-fluorophenyl)sulfone. The crosslinking reaction is exemplified by 1,3-dibromoacetone
O
0
O
0
O
0_o_L,c.-0
0_o_cU.,-
A
B
c,-cLO-~ N
C
O
0
"'~ "
CHICHI__O__C__CH2 I
D
O
~
-c..c,.-,_~_,,÷ L~_s __..~,-c-c,.c..-s-,@ + .
Figure 6 Examples of heterobifunctional crosslinkers: (A) N-succinimidyl 3-maleimidopropionate, (B) N-succinimidyl iodoacetate, (C) 4-maleimidobenzoyl chloride, and (D) ethyl iodoacetimidate. The crosslinking reaction is depicted by N-succinimidyl3-(2-
pyridyldithio)propionate
870
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and crosslinking: S. S. Wong and L.-J. C. Wong
0
'd' "8,
O
o
X 1C I 0
+
=C =S
O P
O
X 1 C --O--N
0
OIN
A
~.._// - - ~ N =C~---N-~/_f ~j t N--OH
0
B
Figure 7 Examples of photoactivatable bifunctional reagents: (A) N-succinimidyl-5-azido-2-nitrobenzoate, and (B) 1-azido-5naphthaleneisothiocyanate
x- c-~-.~
~
- x-c-.-~
0
Figure 8 Crosslinking by activation of a carboxyl group with Nhydroxysuccinimide and cyclohexyl carbodiimide
(~NH
NH | H -~O --C - - N - - @
2 I=
70
H
O .
;f-oN Figure 9 Conjugation with cyanogen bromide. The hydroxyl groups are activated to an active cyclic imidocarbonate which reacts with an amino group of protein
Figure 4. Of the dialdehydes, glutaraldehyde has been extensively utilized. Although it is proposed that the reaction proceeds through a Schiff base, the mechanism is far from clear. 68 Among the dialkylating agents are bis-maleimides, bis-haloacetyl derivatives, di-alkyl halides, and bis-oxiranes. 6° Figure 5 shows the structure of some of these compounds. Although any of the nucleophilic amino acid side chains will react with these crosslinkers, the diacylating agents are generally thought to be amino group-selective, while the dialkylating agents are sulfhydryl-specific. Thiol-specific bis-disulfide crosslinkers have also been synthesized .69 Heterobifunctional crosslinkers contain two different functionalities. These two reactive groups can be any combination of those alkylating and acylating agents mentioned above. For example, one end of the crosslinker may be a disulfide such that it would be sulfhydryl group-specific, and the other end may be an acylating agent for amino group selectivity. Examples of such combinations are shown in Figure 6. Recently, the extremely reactive nitrene- and carbene-generating moieties have been incorporated to provide nondiscriminatory reactions with relatively inert amino acid side chains (Figure 7). 70 There are many other miscellaneous reagents that facilitate crosslinking, particularly protein immobilization. 6°'71-75 These reagents singly or together convert a nonreactive group to a highly reactive intermediate, like the zero-length crosslinkers. The carboxyl groups, for example, may be activated with carbodii-
mide and N-hydroxysuccinimide to form a reactive ester which reacts with the amino group of a protein, as shown in Figure 8. For vicinal diols like those in carbohydrates, e.g., Sepharose, cyanogen bromide may be used to form a highly active cyclic imidocarbonate (Figure 9). This intermediate is highly unstable and undergoes hydrolysis. After isolation from excess reagents, it is coupled to proteins of interest. Cisvicinal diols can also be oxidized with periodate to dialdehydes which form Schiff bases with amino groups. The Schiff bases may be stabilized with reducing agents in a process referred to as reductive alkylation (Figure •0). 76 This method is particularly
OH
~HO
® reducing agent D,
..-®
.,_"_®
Figure 10 Reductive alkylation. The cis-diols are oxidized to dialdehydes followed by Schiff base formation which is stabilized by reduction
E n z y m e M i c r o b . T e c h n o l . , 1992, v o l . 14, N o v e m b e r
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Review O
A.
e.
,,~,-- _ - o -
-
O +
II
EDC
~ P ~ - - C -N H--CH| CHI-NH=I
NH,-CH= CHz-NH,
~1 O O-C--CH=--S-S~CH=-C -O
~ + ~)--NH=
R_N =C==N_R
-
I=
(~ O II H O - C - - C H I - - S _ S ~ CHz ~ C _ N _ _ ~ . ~ )
Figure 11 Introduction of spacers with carbodiimide-mediated reaction. (A) With diamines; (B) with dicarboxylic acids
useful for crosslinking glycoproteins through the carbohydrate moiety. By the same token, polysaccharides may also be used as crosslinkers to couple two or more proteins. 6° Spacers may be introduced between the crosslinked components during the activation process. Diamines and dicarboxylates of different chain lengths can be linked to carboxyl and amino groups, respectively, with carbodiimides, as shown in Figure 11. The ability to introduce different carbon chains provides additional choice to the method. This versatility is further enhanced with the use of bifunctional crosslinking reagents.
More Hydrophobie O
872
O
+ ' L1.-o-c-. O
O
CH:I--O --C --R
O=S--(CH2)= --O --C ~ R
R --O --(CH=) s ~ O - - R
R - O -(CHz)2 -- ~I-(CHz)= - O N R O
,--(ca=).--n
, -~O-(CH=).o-lCH=~.~O--" OH I R --CH= N ( C H ~ C H : I -R
R --(CH=)=--fl
Special features of bifunctional reagents In addition to the reactive head groups of the bifunctional crosslinking reagents, various functional moieties have been incorporated into the backbone of these compounds to increase their adaptability. For example, cleavable bonds have been included to enable the crosslinked species to be separated. These include disulfide bond, vicinal glycol, azo, sulfone, ester, and thioester linkages. 77-83 Reagents, including reducing and oxidizing agents, acids, and bases may be used to cleave these bonds. These reversible crosslinkers are particularly useful for the investigation of protein interactions and for the preparation of immunotoxins. 6° There is little advantage to their use in stabilization of proteins, however. Different degrees of hydrophilicity or hydrophobicity have been incorporated into these compounds, as shown in Figure 12. 60For example, Staros 84incorporated a sulfonate group into the succinimide ring of Nhydroxysuccinimide esters to increase its solubility in aqueous medium. The inclusion of ether-oxygen, hydroxyl group, ester, and amide bonds also increases the solubility in water, while an increase in alkyl chain carbons decreases the hydrophilicity)5 These characteristics may change the hydration sphere of crosslinked proteins, thus causing them to be more or less stable in organic or aqueous media. In addition to changing the degree of hydrophobicity and hydrophilicity, various incorporated entities may also serve as reporter groups. 6° These include spin labels, fluorescence and absorption probes, as well as radioactive moieties (Figure 13). 86-89 Thus, the labeled compounds can be used to change the hydrophobicity and to follow the product. Bifunctional crosslinkers also differ in size in regard to the bulkiness of the molecule, and in length with
More Itydrophilic
Figure 12 Comparison of hydrophobic and hydrophilic crosslinkers. Reagents on the right column are more hydrophilic than those on the left column CH$~, /CH 3 N
+
-
~IH=Cl O==S=OI
+
_
~H2CJ
CH= --O -C ~(CH2) 2 - - N --(CH2)2--C --O --CH=
A I
[~
0
O ~ O O II 0~. / N - O . |1 N--O--C--(CH=)z~C (CH=)= - C - O - - N
o
B 125 I N~
o
OH 0
0
C-O--N
C
O
Figure 13 Bifunctional crosslinkers containing reporter groups: (A) Fluorescent probe, (B) spin label, and (C) radioactive iodide
respect to the distance between the two functional groups. 6° The molecules can be as simple as formaldehyde 9° and as bulky as di[(iodoacetyl)aminomethyl]fluorescein. 9~ The distance between the functional groups can be varied as well. For example, alkyl diimidoesters can be synthesized with different methylene groups, giving rise to different lengths ranging from 5 to 14 A (Figure 14). These molecules are useful for crosslinking different groups on a protein molecule, 92 providing different crosslinked materials.
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and cross~inking: S. S. Wong and L.-J. C. Wong Cross-Linker
Maximum
Distance
3
(,A)
4 5 6
I~H:eCI I~HICI CHI--O--C--CHI -C--O --CHs
7 --(CH2)2 - -
6
8 9
--(CH=)4--
=(CH=)=--
10 ll 12 t0
13 --(CH=)e--
11
14 15
--(OHm)l--
14
16
Figure 14 Di-imidoester crosslinkers of different chain lengths. The maximum distance is that between two coupled groups
20
Application of bifunctional reagents to protein stability
21
As clearly established earlier, chemical crosslinking holds tremendous potential for the stabilization of proteins and enzymes. With the advent of a large number of specific chemical crosslinkers, the application of these compounds for the preparation of stable proteins can be further investigated. Since the 1950s, chemical modification alone has been shown to stabilize enzymes. 93-97Alkylation of the amino groups of a-chymotrypsin, for example, has a stabilizing effect which is dependent on the extent of the reaction. 96 Thus, the crosslinkers, being chemical modification agents, can have the same stabilizing effect by simply reacting with the protein. It has also been shown that increase in hydrophobicity can potentiate its stability. 98 This may be particularly so in hydrophobic solvents. At the same time, hydrophilization has also led to increase in stability, as exemplified by acylation of a-chymotrypsin by pyromellitic dianhydride. 99 As illustrated above, the bifunctional crosslinkers encompass a wide spectrum of hydrophobicity and hydrophilicity. These properties will have to be tested for their efficiency. In addition, the possibility of having different chain lengths for bracing groups of various distances, and the indiscriminating nature of some of these compounds (photoactivatable crosslinkers), have increased the versatility of these reagents. These factors, in addition to the reticulating property, provide tremendous advantages over the simple chemical modification agents for stabilizing proteins and enzymes. A correct choice among these reagents forms the denominator of success.
References 1 2
17 18 19
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
Mozhaev, V. V., Shikshnis, V. A., Torchilin, V. P. and Martinek, K. Biotechnol. Bioeng. 1983, 25, 1937-1948 Trevan, M. D. Biotechnology. John Wiley & Sons, Chichester, England, 1980
44 45
Tatsumoto, K., Oh, K. K., Baker, J. O. and Himmel, M. E. Applied Biochem. Biotechnol. 1990, 20/21, 293-308 Privalov, P. L. Adv. Prot. Chem. 1979, 33, 167-241 Doig, A. J. and Williams, D. H. J. Mol. Biol. 1991,217, 389-398 Prevost, M., Wodak, S. J., Tidor, B. and Karplus, M. Proc. Natl. Acad. Sci. USA 1991, 88, 10880-10884 Sharp, K. A., Nicholls, A., Friedman, R. and Honig, B. Biochemistry 1991, 30, 9686-9697 Alonso, D. O. and Dill, K. A. Biochemistry 1991,30, 5974-5985 Serrano, L., Bycroft, M. and Fersht, A. R. J. Mol. Biol. 1991, 218, 465-475 Akke, M. and Forsen, S. Proteins 1990, 8, 23-29 Jaenicke, R. Eur. J. Biochem. 1991, 202, 715-728 Kellis, J. T., Jr., Nyberg, K., Sail, D. and Fersht, A. R. Nature 1988, 333, 784-786 Zhou, N. E., Kay, C. M. and Hodges, R. S. J. Biol. Chem. 1992, 267, 2664-2670 Chothia, C. and Janin, J. Nature 1975, 256, 705-710 Scheraga, H. A. in The Proteins, Vol. 1, 2nd ed. (Neurath, H., ed) Academic Press, New York, 1963, chap. 6 Tanford, C. The Hydrophobic Effects: Formation o f Micelles and Biological Membranes, 2nd ed. John Wiley & Sons, New York, 1980 Kauzmann, W. Adv. Protein Chem. 1959, 14, 1-63 Stigter, D. and Dill, K. A. Biochemistry 1990, 29, 1262-1271 Schulz, G. E. and Schirmer, R. D. Principles o f Protein Structure. Springer-Verlag, Berlin, 1979 Gilson, M. K., Rashin, A., Fine, R. and Honig, B. J. Mol. Biol. 1985, 183, 503-507 Barlow, D. J. and Thornton, J. M. J. Mol. Biol. 1983, 168, 867-885 Mozhaev, V. V. and Martinek, K. Enzyme Microb. Technol. 1984, 6, 50-65 Mozhaev, V. V. and Martinek, K. Enzyme Microb. Technol. 1982, 2, 299-338 Klibanov, A. M. Adv. Appl. Microbiol. 1983, 29, 1-16 Lumry, R. and Eyring, H. J. Phys. Chem. 1954, 58, ll0-120 Zale, S. E. and Klibanov, A. M. Biotechnol. Bioeng. 1983, 25, 2221-2230 Martinek, K., Klibanov, A. M. and Berezin, I. V. J. SolidPhase Biochem. 1977, 2, 343-355 Klibanov, A. M. Anal. Biochem. 1979, 93, 1-25 Schmid, R. D. Adv. Biochem. Eng. 1979, 12, 41-53 Martinek, K., Klibanov, A. M., Goldmacher, V. S. and Berezin, I. V. Biochim. Biophys. Acta 1977, 485, 1-12 Martinek, K., Klibanov, A. M., Goldmacher, V. S., Tchernysheva, A. V., Mozhaev, V. V., Berezin, I. V. and Glotov, B. O. Biochim. Biophys. Acta 1977, 485, 13-28 Matthews, M. M., Liao, W., Kvalnes-Krick, K. L. and Traut, T. W. Arch. Biochem. Biophys. 1992, 293, 254-263 Mozhaev, V. V., Berezin, I. V. and Martinek, K. CRC Crit. Rev. Biochem. 1988, 23, 235-281 Matsumura, M., Signor, G. and Matthews, B. W. Nature 1989, 16, 291-293 Creighton, T. E. Bioessays 1988, 8, 57-63 Balaji, V. N., Mobasser, A. and Rao, S. N. Biochem. Biophys. Res. Commun. 1989, 160, 109-114 Shaked Z. and Wolfe, S. Methods Enzymol. 1988, 137, 599-615 White, F. L. and Olsen, K. W. Arch. Biochem. Biophys. 1987, 258, 51-57 Yang, T. and Olsen, K. W. Arch. Biochem. Biophys. 1988, 261, 283-290 Yang, T. and Olsen, K. W. Biochem. Biophys. Res. Commun. 1991, 174, 518-523 Tatsumoto, K., Oh, K. K., Baker, J. O. and Himmel, M. E. Appl. Biochem. Biotechnol. 1989, 20/21, 293-308 Torchilin, V. P., Maksimenko, A. V., Klibanov, A. M., Berezin, I. V. and Martinek, K. Biochim. Biophys. Acta 1978, 522, 277-283 Torchilin, V. P., Trubetskoy, V. S. and Martinek, K. J. Mol. Catal. 1983, 19, 291-307 Martinek, K., Klibanov, A. M. and Berezin, I. V. J. Solid Phase Biochem. 1977, 2, 343-352 Wang, P., Hill, T. G., Wartchow, C. A., Huston, M. E., Oehler,
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L. M., Smith, M. B., Bednarski, M. D. and Callstrom, M. R. J. Am. Chem. Soc. 1992, 114, 378-380 Tamura, M., Tamura, T., Burnham, D. N., Uhlinger, D. J. and Lambeth, J. D. Arch. Biochem. Biophys. 1989, 275, 23-32 Wang, D., Wilson, G. and Moore, S. Biochemistry 1976, 15, 660-665 Wang, D. and Moore, S. Biochemistry, 1977, 16, 2937-2942 Wong, S. S., Losiewicz, M. and Wong, L.-J. C. in Biocatalyst Design for Stability and Specificity (Himmel, M. E. and Georgiou, G., eds) Am. Chem. Soc., Washington, D.C., 1992, (in press) Koch-Schmidt, A.-C. and Mosbach, K. Biochemistry 1977, 16, 2105-2109 Daniel, R. M., Cowan, D. A. and Morgan, H. W. Chem. N. Z. 1981, 45, 94-105 Cowan, D. A. and Daniel, R. M. Biochim. Biophys. Acta 1982, 705, 293-305 Gabel, D. Eur. J. Biochem. 1973, 33, 348-412 Demers, A. G. and Wong, S. S. J. Appl. Biochem. 1985, 7, 122-125 Ji, T. H. Methods Enzymol. 1983, 91, 508-609 Han, K.-K., Richard, C. and Delacourte, A. Int. J. Biochem. 1984, 16, 129-144 Gaffney, B. J. Biochim. Biophys. Acta 1985, 822, 289-317 Wawrzynczak, E. J. and Thorpe, P. E. in lmmunoconjugates, Antibody Conjugates in Radioimaging and Therapy of Cancer (Vogel, E.-W., ed) Oxford University Press, New York, 1987, p. 28 Ngo, T. T. Nonisotopic Immunoassay. Plenum Press, New York, 1988 Wong, S. S. Chemistry of Protein Conjugation and Crosslinking. CRC Press, Boca Raton, FL, 1991 Edwards, J. O. and Pearson, R. G. J. Am. Chem. Soc. 1962, 84, 16-26 Kimura, T., Matsueda, R., Nakagawa, Y. and Kaiser, E. T. Anal. Biochem. 1982, 122, 274-282 Patel, R. P., Lopiekes, D. V., Brown, S. P. and Price, S. Biopolymers 1967, 5, 577-582 Miron, T. and Wilchek, M. Methods Enzymol. 1987, 135, 84-90 Monsan, P. and Combes, D. Methods Enzymol. 1988, 137, 584-598 Means, G. E. and Feeney, R. E. Chemical Modification of Proteins. Holden-Day, San Francisco, 1971 Lundblad, R. L. and Noyes, C. M. Chemical Reagents for Protein Modification. Vols. 1 and 2. CRC Press, Boca Raton, FL, 1984 Johnson, T. J. A. in Biocatalyst Design for Stability and Specificity (Himmel, M. E. and Georgiou, G., eds) Am. Chem. Soc., Washington, D.C., 1992 (in press) Bloxham, D. P. and Sharma, R. P. Biochem. J. 1979, 181, 355-366 Bayley, H. and Knowles, J. R. Methods Enzymol. 1977, 46, 69-114 Salmona, M., Saronio, C. and Farattini, S. (eds) lnsolubilized Enzymes. Raven Press, New York, 1974
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
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Johnson, J. C. Immobilized Enzymes, Noyes Data Corp., Park Ridge, NJ, 1979 Scouten, W. H. (ed) Immobilized Enzymes in Solid Phase Biochemistry. Wiley Interscience, New York, 1983 Mosbach, K. (ed) Immobilized Enzymes and Cells, Part D, Methods Enzymol. 1988, 137 Dean, P. D. G., Johnson, W. S. and Middle, F. A. Affinity Chromatography. IRL Press, Washington, DC, 1985 Cabacungan, J. C., Ahmed, A. I. and Feeney, R. E. Anal. Biochem. 1982, 124, 272-278 Traut, R. R., Bollen, A., Sun, T. T., Hershey, J. W. B., Sundberg, J. and Pierce, L. R. Biochemistry 1973, 12, 3266-3273 Webb, J. L. Enzyme and Metabolic lnhibitors. Vol. 2. Academic Press, New York, 1966, p. 729 Smith, R. J., Capaldi, R. A., Muchmore, D. and Dahlquist, F. Biochemistry 1978, 17, 3719-3723 Jaffe, C. L., Lis, H. and Sharon, N. Biochemistry 1980, 19, 4423 -4429 Wold, F. Methods Enzymol. 1972, 25, 623-651 Sato, S. and Nakao, M. J. Biochem. 1981, 90, 1177-1185 Friebel, K., Huth, H., Jany, K. D. and Trummer, W. E. Z. Physiol. Chem. 1981, 362, 421-428 Staros, J. V. Biochemistry 1982, 21, 3950-3955 Fasold, H., B~iumert, H. and Fink, G. in Protein Cross-linking: Biochemical and Molecular Aspects (Friedman, M., ed) Plenum Press, New York, 1976, p. 207 Gonzalez-Ros, J. M., Farach, M. C. and Martinez-Carrion, M. Biochemistry 1983, 22, 3807-3811 Gaffney, B. J., Willingham, G. L. and Schepp, R. S. Biochemistry 1983, 22, 881-892 Ji, T. H. and Ji, I. Anal. Biochem. 1982, 121, 286-289 Sigrist, H., Allegrini, P. R., Kempf, C., Schnippering, C. and Zahler, P. Eur. J. Biochem. 1982, 125, 197-201 Hopwood, D. Histochemie 1969, 17, 151-161 Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, OR, 1990, p. 22 Ji, T. H. J. Biol. Chem. 1974, 249, 7841-7847 Sri Ram, J., Terminiello, L., Bier, M. and Nord, F. F. Arch. Biochem. Biophys. 1954, 52, 464 Torchilin, V. P. and Martinek, K. Enzyme Microb. Technol, 1979, 74, 1-19 Branner-Yorgensen, S. Biochem. Soc. Trans. 1983, 11, 20-31 Torchilin, V. P., Maksimenko, A. V., Smirnov, V. N., Berezin, I. V. and Martinek, K. Biochim. Biophys. Acta 1979, 567, 1-I1 Urabe, I., Yamanoto, M., Yamada, Y. and Okada, H. Biochim. Biophys. Acta 1978, 524, 435-441 Shatsky, M. A., Ho, H. C. and Wang, J. H. Biochim. Biophys. Acta 1973, 303, 298-307 Mozahev, V. V., Siksnis, V. A., Melik-Nubarov, N. S., Galkantaite, N. Z., Denis, G. J., Butkus, E. P., Zaslavsky, B. Y., Mestechkina, N. M. and Martinek, K. Eur. J. Biochem. 1988, 173, 147-154
The technique of chemical crosslinking has been used to enhance the stability of proteins and enzymes. In this procedure, the molecule is braced with chemical crosslinks either intramolecularly or intermolecularly to another species to reinforce its active structure. Various chemicals have been used for this purpose. The bifunctional reagents are the most prominent. These compounds are derived from groupspecific reagents and may be classified into homobifunctional, heterobifunctional, and zero-length crosslinkers. Different physical and chemical characteristics have been incorporated into these chemicals. Their versatility holds great potential in preparing chemically, thermally, and mechanically stable proteins and enzymes for industrial applications.
Keywords: Protein stability; chemical crosslinking, crosslinking reagents; enzyme activity; bifunctional reagents
Introduction The stability of proteins and enzymes is a major concern in their industrial applications. Biocatalysts with high thermostability will have a prolonged viability, t A lengthened operational stability will reduce the need for frequent enzyme replacement and thus decrease the cost of enzyme preparations, which can be rather expensive. 2 These thermostable enzymes will enable the acceleration of chemical reaction rates at a higher temperature. According to Van't Hoff's rule, an increase of temperature by 10°C will raise the rate of chemical reactions by two- to threefold. In cases where several reaction steps are required, a thermostable biocatalyst will enable the process to continue at elevated temperatures without lowering the heat. 3 Reactions occurring at higher temperatures will also shift the thermodynamic equilibrium. For endothermic processes, product formation will be favored at elevated temperatures, as stated by Le Chatelie's principle. Reactions at high temperatures also provide a simple termination procedure by cooling. Not only high thermostability is desirable, but also stability in organic solvents, in extreme pH values and pressure, and towards mechani-
Address reprint requests to Dr. S. S. Wong at the Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030 Accepted for publication 14 June 1992
866
cal disturbances is required for organic synthesis, chemical analysis, isolation, and purification of chemicals, in therapeutics and diagnostics, and in the study of protein structures and functions. These and other advantages have prompted a persistent search for methods to prepare stable proteins and enzymes.
Chemical basis of protein stability Recent interests in structure-stability relationships in proteins have led to the calculations of free energies contributed by various structural determinants. Numerous articles and reviews have been published. 4-H A brief summation is included here for the understanding of the approaches undertaken to prepare stable proteins and enzymes. Hydrophobicity has always been considered one of the major factors of protein integrity. 12.13Hydrophobic side chains are usually buried inside the protein molecules to induce a tight packing. For proteins that associate with other components, hydrophobic clusters are found localized on the surface of the protein molecules to induce solitary contacts with other proteins, with lipids, or with other hydrophobic entities. 14 This shielding of nonpolar groups from interaction with water in the medium reduces the unfavorable entropy of the system and thus increases protein stability.15-~7 In addition to hydrophobic forces, hydrogen bonding, salt bridges, dipole-dipole, and other electrostatic interactions also contribute to protein stability. 18 Hydrogen bonds play an important role in the maintenance of
Enzyme Microb. Technol., 1992, vol. 14, November
© 1992 Butterworth-Heinemann
Stability and crosslinking: S. S. Wong and L.-J. C. Wong secondary structures of proteins, i.e., o~-helixes, /3sheets, etc. They also provide an interaction of polar groups of protein and water at the protein-water interface to form a hydration sphere of the molecule. 14While the energy contribution of dipole-dipole interactions is small, the large number of such interactions in proteins makes it significant.19 On the other hand, the number of salt bridges is small, but their energy contribution is large. 2°,21 It is the sum of these various forces, together with the binding of metal ions, substrates, cofactors, and other low-molecular-weight ligands, that maintains the structural integrity of a functional protein or enzyme. 22 Although the molecular mechanism of protein inactivation may involve several conformational changes leading to the unfolding of the molecule,23'24 the overall physical process may be represented simply by two steps: N ~ I---~ D where N, I, and D are native, intermediate, and denatured forms of the protein, respectively. 23-26 The first step involves reversible conformational changes, followed by a second step of irreversible unfolding by which an enzyme loses its activity. The approach to stabilizing proteins and enzymes is to prevent irreversible unfolding from occurring. 27-31This can be achieved by several means.
Methods of stabilization Many practical methods have evolved to preserve the integrity and activity of the native proteins. Based on thermodynamic reasoning, proteins were invariably kept at low temperatures to prolong their longevity. Although many enzymes and proteins can be stored in this manner for an extended period of time, low temperatures are not preferable in many industrial operations. Furthermore, some proteins are cold-sensitive and may be more stable at ambient temperatures. In an attempt to mimic the microenvironment of the proteins in the cell, various substances have been added to interact with isolated proteins. Glycerol, ethylene glycol, and detergents have been used to preserve enzyme activities after isolation, particularly enabling the proteins to be kept at very low temperatures without being frozen. 32 Other substances, such as carbohydrates (e.g., sucrose) and proteins (e.g., albumin), have also been used to stabilize proteins of interest. While these methods have preserved the biological activities to various extents, the procedure introduces foreign substances, which may not be desirable in many applications. A technique to increase the contributions of various forces to the free energy of stabilization is protein engineering, involving site-specific mutagenesis and cloning of thermostable molecules whose structures mimic that of proteins isolated from thermophilic organisms.33 This method seems to be appealing, but tremendous difficulties associated with protein characterization, including three-dimensional structural analysis, will have
to be resolved before site-specific mutagenesis can be defined. Another approach to stabilizing proteins and enzymes is to strengthen the compact structure of the molecule so that denaturation will not occur. This may be accomplished by chemical crosslinking. It has been demonstrated that intramolecular disulfide bonds contribute significant stabilization energy. Doig and Williams 5'34'35 have argued that disulfide bonds destabilize folded structures entropically, but stabilize them enthalpically to a greater extent, resulting in an overall stabilization of the molecule. Balaji et al. 36 have used x-ray crystal structure data to define geometric parameters in serine proteases and subtilisin to introduce disulfide bridges which lead to thermal stability. Like disulfide bonds, chemical crosslinking reagents have been used to brace proteins intramolecularly and to conjugate them intermolecularly to other molecules, including solid supports. This technique has been demonstrated to greatly enhance the stability of proteins.
Stability effected by chemical crosslinking If the molecular basis of inactivation is a reflection of the disruption of the secondary and tertiary structure of a protein, the active conformation may be reinforced with chemical crosslinking reagents. As depicted schematically in Figure 1, chemical crosslinks will reticulate the molecule to diminish the polypeptide entropy, which will decrease the rate of denaturation. With multiple attachments, the molecule is held in its original active structure. The braces can be achieved by both intra- and intermolecular crosslinking. Many studies have shown that intramolecular crosslinking is effective in protein stabilization. Shaked and W o l f e 37 have
CTV Figure 1 Stability by intra- and intermolecular crosslinking. (A) Intramolecular crosslinks reticulate the molecule to reinforce the structure. Multipoint intermolecular crosslinking (B) to a solid support and (C) to a soluble entity has a similar effect
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Review used dimethyl suberimidate and dimethyl adipimidate to crosslink catalase. The process removed 44 of the reactive amino groups and showed impressive stability towards glucosone and temperature. Olsen e t a / . 38-4° have crosslinked hemoglobin with bis(3,5-dibromosalicyl) fumarate. A single crosslink between Lys82/31 and Lys82/32 increased the denaturation transition temperature of methemoglobin A from 40.7 to 57. I°C. For cyanomethemoglobin the transition increased from 55.5 to 67.5°C. With deoxyhemoglobin, a crosslink between Lys99al and Lys99a2 was obtained. The a crosslinked hemoglobin has the same stability as the/3 crosslink, with a gain in transition temperature of 17°C. Since the amino acid residues modifiable by crosslinking reagents are distributed randomly in a protein, their positions in the three-dimensional structure differ in space and microenvironment. Different crosslinking reagents may vary significantly in their ability to confer stability. Tatsumoto et al. 41 have used a series of bifunctional chemical modification reagents of different chain lengths to crosslink amyloglucosidase. Several of the diimidoesters were shown to more than double the half-life of this enzyme at 65°C, but dimethylglutarimidate provided the longest half-life. Torchilin et al. 42 have reported that the degree of increased thermostability of a-chymotrypsin depends on the length of carbon chain in the intramolecular crosslinks. The highest stabilizing effect is produced by carbodiimide-activated enzyme with 1,4-tetramethylenediamine. Similarly, intramolecularly crosslinked glyceraldehyde-3phosphate dehydrogenase is more stable at 60°C than native enzyme, and the degree of stabilization is dependent on the chain length of the bifunctional reagent u s e d . 43 Maximal thermostabilization was found when succinic acid was used in the carbodiimide-activated enzyme. Multipoint fixation of enzymes to solid supports or soluble polymers has been shown to increase the resistance of enzymes and proteins to high temperatures and denaturants such as u r e a . 28'33'44'45 Crosslinking of superoxide-generating respiratory burst oxidase in neutrophil plasma membranes with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, has significantly enhanced enzyme stability towards heat, high salt, and detergent. 46 The crosslinked enzyme has an increased half-life from 2 to over 20 min at 37°C, and is completely active in 0.1 M KCI and 0.4% Triton X-100, which have inactivating effects on the oxidase. Intermolecular crosslinking of enzymes of interest to other soluble proteins is also beneficial. Such an indication was first demonstrated by ribonuclease when it was crosslinked with dimethyl suberimidate to itself to form a dimer or to polyspermine to yield a heteroconjugate. 47'48 The polysperimine-ribonuclease conjugate was 115 times more active towards poly(A) • poly(U), and 176 times more active towards poly(I). poly(C) than the parent enzyme. 48 Further studies revealed that when horseradish peroxidase is crosslinked to immunoglobulin G or Jacalin, the energy of inactivation of the enzyme increased from 35 to 51 and 43 Kcal
868
mo1-1, respectively. 49 Recently, Wang et al. 45 have synthesized a series of aminoglucose-based carbohydrate polymers which have been demonstrated to stabilize proteases in organic solvents and elevated temperatures. Immobilization of molecules to solid supports has long been recognized to confer stability. 27-29A severalhundred- to a thousandfold decrease in both the reversible and irreversible thermoinactivation of enzymes has been observed. ~.30,3~.50Most strikingly, highly heat-stable thermophilic asparaginase and caldolysin can be additionally stabilized 10 times by immobilization. 5~'52 Stability towards denaturants such as urea has also been noted. 53 Demers and W o n g 54 have demonstrated that immobilization can also increase the mechanical stability, as shown for galactosyltransferase. Thus, there are ample examples to illustrate that chemical crosslinking, either inter- or intramolecular, provides a means of preparing stable enzymes and proteins.
General characteristics of crosslinking reagents Chemical crosslinking of proteins and enzymes can be most easily achieved with bifunctional compounds in comparison to monofunctional reagents. 5°,55-6° The bifunctional reagents are essentially chemical modification agents and may be classified into zero-length, homobifunctional, and heterobifunctional crosslinkers. These chemicals react with nucleophilic side chains of amino acids, such as the sulfhydryl group of cysteine, the amino groups of lysine and the N-terminal amino acids, the carboxyl groups of aspartic and glutamic acids and the C-terminal amino acids, the imidazolyl group of histidine, and the thioether group of methionine. The specificity of these chemicals for a specific amino acid side chain depends on the relative reactivity of the nucleophile. Since the nucleophilicity is a function of the electronic structure, the P K a , and the microenvironment, the reactivity of an amino acid side chain is generally not specific, and several side chains may react with the same bifunctional reagent. 6° In general, however, the thiolate ion is the most nucleophilic. 61 Thus the sulfhydryl groups of proteins will react with most of the crosslinkers at alkaline pH. For example, a-haloacetyl compounds and N-maleimido derivatives are generally considered as sulfhydryl-selective. A different set of reagents that are thiol-specific are disulfide and mercurial compounds. Disulfide compounds react through a disulfide interchange reaction, as shown in Figure 2. 62
In proteins where the free sulfhydryl moiety is not available, the amino group becomes the major target of reaction. Also, for proteins with high surface amino group content, the competition for the reagent may become favorable. In addition, thioacyl esters, as a result of acylation, are susceptible to hydrolysis. Thus, acylating agents are in general considered amino groupdirecting. Of the crosslinking reagents, the zero-length crosslinkers induce direct joining of two chemical compo-
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and crosslinking:
@s-cl
_!-.N-
S-SONO,
NOz
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ai,-s
-s
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coon A
0
C
Figure 2 Disulfide interchange reaction. The free thiolate ion displaces another thiol from a disulfide compound. Examples of disulfide reagents are: (A) 5,5’-dithiobis-(2nitrobenzoic acid); (5) 4,4’-dithiopyridine, and (C) methyl-3-nitro-2-pyridyl disulfide
nents without the introduction of any extrinsic material. This is in contrast to homo- and heterobifunctional reagents, where a spacer is incorporated between the two crosslinked groups. Examples of zero-length crosslinkers are carbodiimides, isoxazolium derivatives, chloroformates, and carbonyldiimidazole,63-66
S. S. Wong and L.-J. C. Wong
which couple carboxyl and amino groups, and cupric di( 1, IO-phenanthroline) and 2,2’-dipyridyldisulfide, which form disulfide bonds. These reagents react by activating one of the moieties to form an active intermediate, which then reacts with a second component, as shown in Figure 3. The other classes of crosslinkers contain two reactive functionalities which will react with amino acid side chains, thus bringing the two components together. The reactive groups are located at the two ends of the molecule, connected by a backbone which may be designed to contain specific characteristics. Because the most reactive amino acid side chains susceptible of modification are nucleophiles, acylating and alkylating agents are incorporated into these head groups.S*67 When a crosslinker contains two identical reactive groups, it is referred to as a homobifunctional reagent. Examples of diacylating agents include bisimidoesters, bis-succinimidyl derivatives, di-isocyanates, di-isothiocyanates, di-sulfonyl halides, bis-nitrophenyl esters, dialdehydes, and diacylazides.N A few common representative compounds are shown in
_oP,
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Figure 3 Examples and mechanism carbonyl diimidazole
of action of zero-length
crosslinkers:
(A) ethyl chloroformate,
Enzyme Microb. Technol.,
(6) a carbodiimide,
and (C) l,l’-
1992, vol. 14, November
889
Review O
O l'
N
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A
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C
D
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"~
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Figure 4 Examples of diacylating homobifunctional reagents: (A) disuccinimidyl suberate, (B) dimethyl malonimidate, (C) 1,4-dicyanatobenzene, and (D) p-phenylene-diisothiocyanate. The mechanism for crosslinking is illustrated by phenol-2,4-disulfonyl chloride
O
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0
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Figure 5 Examples of dialkylating homobifunctional reagents: (A) N,N'-methylenebismaleimide, (B) e,~'-diiodo-p-xylene sulfonic acid, (C) di(2-chloroethyl)sulfone, and (D) bis(3-nitro-4-fluorophenyl)sulfone. The crosslinking reaction is exemplified by 1,3-dibromoacetone
O
0
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B
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D
O
~
-c..c,.-,_~_,,÷ L~_s __..~,-c-c,.c..-s-,@ + .
Figure 6 Examples of heterobifunctional crosslinkers: (A) N-succinimidyl 3-maleimidopropionate, (B) N-succinimidyl iodoacetate, (C) 4-maleimidobenzoyl chloride, and (D) ethyl iodoacetimidate. The crosslinking reaction is depicted by N-succinimidyl3-(2-
pyridyldithio)propionate
870
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and crosslinking: S. S. Wong and L.-J. C. Wong
0
'd' "8,
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o
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+
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O
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0
B
Figure 7 Examples of photoactivatable bifunctional reagents: (A) N-succinimidyl-5-azido-2-nitrobenzoate, and (B) 1-azido-5naphthaleneisothiocyanate
x- c-~-.~
~
- x-c-.-~
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Figure 8 Crosslinking by activation of a carboxyl group with Nhydroxysuccinimide and cyclohexyl carbodiimide
(~NH
NH | H -~O --C - - N - - @
2 I=
70
H
O .
;f-oN Figure 9 Conjugation with cyanogen bromide. The hydroxyl groups are activated to an active cyclic imidocarbonate which reacts with an amino group of protein
Figure 4. Of the dialdehydes, glutaraldehyde has been extensively utilized. Although it is proposed that the reaction proceeds through a Schiff base, the mechanism is far from clear. 68 Among the dialkylating agents are bis-maleimides, bis-haloacetyl derivatives, di-alkyl halides, and bis-oxiranes. 6° Figure 5 shows the structure of some of these compounds. Although any of the nucleophilic amino acid side chains will react with these crosslinkers, the diacylating agents are generally thought to be amino group-selective, while the dialkylating agents are sulfhydryl-specific. Thiol-specific bis-disulfide crosslinkers have also been synthesized .69 Heterobifunctional crosslinkers contain two different functionalities. These two reactive groups can be any combination of those alkylating and acylating agents mentioned above. For example, one end of the crosslinker may be a disulfide such that it would be sulfhydryl group-specific, and the other end may be an acylating agent for amino group selectivity. Examples of such combinations are shown in Figure 6. Recently, the extremely reactive nitrene- and carbene-generating moieties have been incorporated to provide nondiscriminatory reactions with relatively inert amino acid side chains (Figure 7). 70 There are many other miscellaneous reagents that facilitate crosslinking, particularly protein immobilization. 6°'71-75 These reagents singly or together convert a nonreactive group to a highly reactive intermediate, like the zero-length crosslinkers. The carboxyl groups, for example, may be activated with carbodii-
mide and N-hydroxysuccinimide to form a reactive ester which reacts with the amino group of a protein, as shown in Figure 8. For vicinal diols like those in carbohydrates, e.g., Sepharose, cyanogen bromide may be used to form a highly active cyclic imidocarbonate (Figure 9). This intermediate is highly unstable and undergoes hydrolysis. After isolation from excess reagents, it is coupled to proteins of interest. Cisvicinal diols can also be oxidized with periodate to dialdehydes which form Schiff bases with amino groups. The Schiff bases may be stabilized with reducing agents in a process referred to as reductive alkylation (Figure •0). 76 This method is particularly
OH
~HO
® reducing agent D,
..-®
.,_"_®
Figure 10 Reductive alkylation. The cis-diols are oxidized to dialdehydes followed by Schiff base formation which is stabilized by reduction
E n z y m e M i c r o b . T e c h n o l . , 1992, v o l . 14, N o v e m b e r
871
Review O
A.
e.
,,~,-- _ - o -
-
O +
II
EDC
~ P ~ - - C -N H--CH| CHI-NH=I
NH,-CH= CHz-NH,
~1 O O-C--CH=--S-S~CH=-C -O
~ + ~)--NH=
R_N =C==N_R
-
I=
(~ O II H O - C - - C H I - - S _ S ~ CHz ~ C _ N _ _ ~ . ~ )
Figure 11 Introduction of spacers with carbodiimide-mediated reaction. (A) With diamines; (B) with dicarboxylic acids
useful for crosslinking glycoproteins through the carbohydrate moiety. By the same token, polysaccharides may also be used as crosslinkers to couple two or more proteins. 6° Spacers may be introduced between the crosslinked components during the activation process. Diamines and dicarboxylates of different chain lengths can be linked to carboxyl and amino groups, respectively, with carbodiimides, as shown in Figure 11. The ability to introduce different carbon chains provides additional choice to the method. This versatility is further enhanced with the use of bifunctional crosslinking reagents.
More Hydrophobie O
872
O
+ ' L1.-o-c-. O
O
CH:I--O --C --R
O=S--(CH2)= --O --C ~ R
R --O --(CH=) s ~ O - - R
R - O -(CHz)2 -- ~I-(CHz)= - O N R O
,--(ca=).--n
, -~O-(CH=).o-lCH=~.~O--" OH I R --CH= N ( C H ~ C H : I -R
R --(CH=)=--fl
Special features of bifunctional reagents In addition to the reactive head groups of the bifunctional crosslinking reagents, various functional moieties have been incorporated into the backbone of these compounds to increase their adaptability. For example, cleavable bonds have been included to enable the crosslinked species to be separated. These include disulfide bond, vicinal glycol, azo, sulfone, ester, and thioester linkages. 77-83 Reagents, including reducing and oxidizing agents, acids, and bases may be used to cleave these bonds. These reversible crosslinkers are particularly useful for the investigation of protein interactions and for the preparation of immunotoxins. 6° There is little advantage to their use in stabilization of proteins, however. Different degrees of hydrophilicity or hydrophobicity have been incorporated into these compounds, as shown in Figure 12. 60For example, Staros 84incorporated a sulfonate group into the succinimide ring of Nhydroxysuccinimide esters to increase its solubility in aqueous medium. The inclusion of ether-oxygen, hydroxyl group, ester, and amide bonds also increases the solubility in water, while an increase in alkyl chain carbons decreases the hydrophilicity)5 These characteristics may change the hydration sphere of crosslinked proteins, thus causing them to be more or less stable in organic or aqueous media. In addition to changing the degree of hydrophobicity and hydrophilicity, various incorporated entities may also serve as reporter groups. 6° These include spin labels, fluorescence and absorption probes, as well as radioactive moieties (Figure 13). 86-89 Thus, the labeled compounds can be used to change the hydrophobicity and to follow the product. Bifunctional crosslinkers also differ in size in regard to the bulkiness of the molecule, and in length with
More Itydrophilic
Figure 12 Comparison of hydrophobic and hydrophilic crosslinkers. Reagents on the right column are more hydrophilic than those on the left column CH$~, /CH 3 N
+
-
~IH=Cl O==S=OI
+
_
~H2CJ
CH= --O -C ~(CH2) 2 - - N --(CH2)2--C --O --CH=
A I
[~
0
O ~ O O II 0~. / N - O . |1 N--O--C--(CH=)z~C (CH=)= - C - O - - N
o
B 125 I N~
o
OH 0
0
C-O--N
C
O
Figure 13 Bifunctional crosslinkers containing reporter groups: (A) Fluorescent probe, (B) spin label, and (C) radioactive iodide
respect to the distance between the two functional groups. 6° The molecules can be as simple as formaldehyde 9° and as bulky as di[(iodoacetyl)aminomethyl]fluorescein. 9~ The distance between the functional groups can be varied as well. For example, alkyl diimidoesters can be synthesized with different methylene groups, giving rise to different lengths ranging from 5 to 14 A (Figure 14). These molecules are useful for crosslinking different groups on a protein molecule, 92 providing different crosslinked materials.
Enzyme Microb. Technol., 1992, vol. 14, November
Stability and cross~inking: S. S. Wong and L.-J. C. Wong Cross-Linker
Maximum
Distance
3
(,A)
4 5 6
I~H:eCI I~HICI CHI--O--C--CHI -C--O --CHs
7 --(CH2)2 - -
6
8 9
--(CH=)4--
=(CH=)=--
10 ll 12 t0
13 --(CH=)e--
11
14 15
--(OHm)l--
14
16
Figure 14 Di-imidoester crosslinkers of different chain lengths. The maximum distance is that between two coupled groups
20
Application of bifunctional reagents to protein stability
21
As clearly established earlier, chemical crosslinking holds tremendous potential for the stabilization of proteins and enzymes. With the advent of a large number of specific chemical crosslinkers, the application of these compounds for the preparation of stable proteins can be further investigated. Since the 1950s, chemical modification alone has been shown to stabilize enzymes. 93-97Alkylation of the amino groups of a-chymotrypsin, for example, has a stabilizing effect which is dependent on the extent of the reaction. 96 Thus, the crosslinkers, being chemical modification agents, can have the same stabilizing effect by simply reacting with the protein. It has also been shown that increase in hydrophobicity can potentiate its stability. 98 This may be particularly so in hydrophobic solvents. At the same time, hydrophilization has also led to increase in stability, as exemplified by acylation of a-chymotrypsin by pyromellitic dianhydride. 99 As illustrated above, the bifunctional crosslinkers encompass a wide spectrum of hydrophobicity and hydrophilicity. These properties will have to be tested for their efficiency. In addition, the possibility of having different chain lengths for bracing groups of various distances, and the indiscriminating nature of some of these compounds (photoactivatable crosslinkers), have increased the versatility of these reagents. These factors, in addition to the reticulating property, provide tremendous advantages over the simple chemical modification agents for stabilizing proteins and enzymes. A correct choice among these reagents forms the denominator of success.
References 1 2
17 18 19
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
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