The chemical modification of {alpha}-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water–organic media

A.A. Vinogradov1,2, E.V. Kudryashova3, V.Ya. Grinberg4, N.V. Grinberg4, T.V. Burova4 and A.V. Levashov3

1 A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov str., 119991 Moscow, 3 Department of Chemical Enzymology, M.V. Lomonosov Moscow State University, Vorobievy gory, 117899 Moscow and 4 N.M. Emmanuel Institute of Biochemical Physics, 4 Kosygina str.,117977 Moscow, Russia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We considered {alpha}-chymotrypsin (CT) in homogeneous water–organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water–ethanol and water–DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water–ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.

Keywords: {alpha}-chymotrypsin/chemical modification/kinetics/stabilization/water—organic mixtures


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently there has been impressive progress in biocatalyst design tools, such as directed evolution (Arnold et al., 1999). Nevertheless, understanding structure–function relationships is still one of the challenging tasks in protein science. It is well known that even minor structural changes may cause dramatic changes of protein properties. The protein surface is primarily responsible for interaction with the surrounding solvent. Thus, the properties of the enzyme surface may be crucial for maintaining its catalytically active conformation, particularly in non-natural environments (non-water and water–organic solvents). The possibility of enzymatic reactions in such media has greatly increased the industrial use of enzymes (Khmelnitsky and Rich, 1999Go). Therefore, it is interesting from both theoretical and practical points of view to determine the relationships between enzyme surface properties and enzyme stability in water–organic solvent media.

Biocatalysis in non-aqeous media has been the subject of numerous studies (Oldfield, 1994Go; Tuena de Gomez-Puyou and Gomez-Puyou, 1998Go; Halling, 2000Go; Klibanov, 2001Go). Some enzyme suspensions in non-polar water-immiscible organic solvents were shown be to very stable (Zaks and Klibanov, 1985Go). It has been suggested that in more polar solvents, water molecules, essential for stabilizing the native conformation, are stripped from the protein surface (Zaks and Klibanov, 1984Go). Several approaches have been proposed to make water molecules bind more tightly or to immobilize the protein in hydrophilic matrices (Khmelnitsky and Rich, 1999Go).

Enzymes dissolved in polar organic solvents or in homogeneous water–organic media usually lose their native conformation and catalytic activity. It is generally believed that the introduction of additional hydrophilic groups on the protein surface may improve its stability in such systems owing to the enhanced ability of the hydrophilized surface to keep the hydration shell and form additional electrostatic interactions, hydrogen bonds or salt bridges (Khmelnitsky et al., 1991Go; Mozhaev et al., 1996Go). On the other hand, hydrophobic groups, if introduced near the hydrophobic region on protein surface, may also contribute to protein stabilization (Lee and Richards, 1971Go; Chotia, 1984Go). Arnold and co-workers suggested that the replacement of disordered, uncompensated surface charge with hydrophobic residues using site-directed mutagenesis may dramatically improve enzyme stability (Arnold, 1988Go; Martinez and Arnold, 1991Go). Crambin, a small hydrophobic protein, is an amazing demonstration of natural protein design for non-aqueous media: it is soluble and retains its native structure at very high concentrations of polar organic solvents (De Marco, 1981). Crambin is not soluble in aqueous solutions, but has water-soluble homologs with similarly folded structure (Teeter et al., 1981Go). In comparison with these homologs, crambin possesses amino acid substitutions, removing hydrophilic side chains: lysines, arginines and asparagines are almost excluded in crambin.

In recent work, we have performed hydrophobization/ hydrophilization via chemical modification of {alpha}-chymotrypsin surface amino groups. The aim was to understand how both types of modification affect enzyme activity, structure and stability.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Covalent modification of {alpha}-chymotrypsin

Acetylation of {alpha}-chymotrypsin (CT) (Sigma, EC 3.4.21.1) was carried out with pyromellitic anhydride (Aldrich) and succinic anhydride (Reanal) as described previously (Mozhaev et al., 1988Go).

Reductive alkylation with glyceric, pentylic, isobutyric and acetic aldehydes (Reanal) was carried out according to Mozhaev et al. (Mozhaev et al., 1992Go). Modification with a mixture of glyceric and pentyl aldehydes was made in exactly the same way (a 100 molar excess of each aldehyde with respect to the enzyme was taken).

Determination of the degree of modification

The degree of CT modification was calculated from the number of amino groups in it which reacted with trinitrobenzenesulfonic acid (TNBS) as compared with the unmodified enzyme (Fields, 1971Go).

Titration of {alpha}-chymotrypsin active sites

The concentration of active sites in all CT samples was determined with N-trans-cinnamoylimidazole (Sigma) as titration reagent (Schonbaum et al., 1961Go).

Other procedures

Inhibition with phenylmethanesulfonyl fluoride (PMSF) was performed as described (Fahrney and Gold, 1963Go).

The enzymatic activity of {alpha}-chymotrypsin in binary water–organic mixtures was determined with N-benzoyl-L-tyrosine-p-nitroanilide (BTNA) (Sigma) and with 4-methylumbelliferyl-p-trimethylammonium cinnamate chloride (MUTMAC) as described previously (Kudryashova et al., 1997Go).

Calculations of hydrophobicity of modifiers and of change of hydrophobicity of modified CT were made according to Mozhaev et al. (Mozhaev et al., 1992Go).

Differential scanning calorimetry (DSC) measurements were made essentially as described elsewhere (Grinberg et al., 2000Go). Protein solutions for calorimetric measurements were dialyzed against corresponding media for at least 5 h at 4°C. Buffer solution was 20 mM MOPS, pH 7.45. The protein concentration after dialysis was determined spectrophotometrically using the extinction coefficient E280 = 50 000 M–1 cm–1 (Volini and Tobias, 1969Go). The concentration of protein solutions used for calorimetric measurements was 0.5–1.2 mg/ml. DSC measurements were carried out with a DASM-4 adiabatic differential scanning microcalorimeter (Biopribor, Puschino, Russia) at a heating rate of 1°C/per min and at extra pressure 1 atm. Primary data processing was performed using NAIRTA software (Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia).

A quantitative thermodynamic analysis should be based on equilibrium studies. Nevertheless, in some cases one can use this analysis for practically irreversible processes (Privalov and Potekhin, 1986Go). The thermal denaturation of CT is known to be irreversible. To minimize the interference of irreversible denaturation, all measurements were made under the same conditions. We aimed to compare the calorimetric parameters of modified and native samples of CT.

Circular dichroism experiments were carried out with a Jobin Yvon Mark V spectrometer at 25°C with quartz cells of pathlength 1 mm in the far-UV region (200–260 nm). The protein concentrations were 0.1–0.2 mg/ml. The secondary structure percentage predictions were made using CDNN software (http://bioinformatik.biochemtech.uni-halle.de/cdnn).

Polyacrylamide gel electrophoresis was performed under denaturing conditions according to Laemmli (Laemmli, 1970Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enzymatic activity

We found that both surface hydrophilization and hydrophobization enhance protein stability against denaturation in water–organic mixtures. This effect was expressed in (i) broadening of the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value and (ii) the increase in the organic cosolvent concentration range at which the residual enzyme activity is observed.

In water–organic solvent mixtures native (non-modified) CT is relatively unstable (Mozhaev et al., 1989Go; Kudryashova et al., 1994Go; Gladilin et al., 1995Go). It is almost inactive at 50% (v/v) ethanol (EtOH), which is in agreement with data from other groups (Sato et al., 2000Go); in 40% (v/v) dimethylformamide (DMF) CT is completely inactive owing to denaturation. At moderate organic cosolvent concentrations (up to 20%, v/v) native CT is more active than in a water environment (Figures 1 and 2GoGo). Further, the catalytic activity is gradually decreased to complete inactivation.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Catalytic activity of some CT samples in water–DMF mixtures. {blacklozenge}, Native CT; {blacktriangleup}, CT modified with pyromellitic anhydride, degree of modification 11 (pyr11CT); *, CT modified with glyceric aldehyde, degree of modification 13 (glyc13CT); {blacksquare}, CT modified with pentyl aldehyde, degree of modification 11 (pent11CT).

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Catalytic activity of some CT samples in water–ethanol mixtures. Symbols as in Figure 1Go.

 
Native CT was modified with several hydrophilic reagents (degrees of modification are given in parentheses): succinic anhydride (3, 6), pyromellitic anhydride (2, 8, 11, 15) and glyceric aldehyde (2, 6, 13) (Table IGo). A substantial stabilization effect was achieved for pyromellitic and glyceric anhydrides. At higher degrees of modification this effect was greater (Figures 1 and 2GoGo). The stabilization effect of succinic anhydride was less pronounced. Samples modified with glyceric aldehyde (glycCT) were found to be more resistant to aggregation in water–ethanol mixtures: precipitation of glycCT was observed at 80% (v/v) ethanol, whereas native CT precipitated at 40%.


View this table:
[in this window]
[in a new window]
 
Table I. The increments of hydrophobicity ({Delta}{Delta}G) introduced by the modifications

 
For hydrophobic modification of CT we used pentylic aldehyde (degree of modification 3 and 11), isobutyric aldehyde (degree of modification 6 and 7) and acetic aldehyde (degree of modification 5). Modifications with pentylic (pentCT) and isobutyric aldehydes (ibutCT) led to substantial stabilization against denaturation in water–organic mixtures (Figures 1 and 2GoGo). This case is particularly interesting, because the replacement of a surface charged amino group with a hydrophobic chain is not usually expected to stabilize the protein. Moreover, the resistance of the samples against aggregation in water–ethanol mixtures was enhanced in comparison with native CT: ibutCT and pentCT formed true solutions at 80% (v/v) ethanol. Modification with acetic aldehyde did not have much effect on the CT behavior in water–organic solvents.

Normally, not more than 20% of CT active sites were lost in the course of modification. The catalytic properties of modified samples were not dramatically different from native enzyme owing to several experimental facts. (1) The hydrolysis of two substrates differing in the rate-limiting step, BTNA and MUTMAC, was investigated. The rate-limiting step for BTNA is acyl-enzyme formation (West et al., 1990Go) and for MUTMAC it is deacylation (Maurel, 1978Go). The catalytic activity of modified samples in aqueous solution does not change from native CT by more than 50%, taking into account the percentage of the active sites. In water–organic media the modified samples demonstrate similar trends to the native enzyme, but possess higher stability (Figures 1 and 2GoGo). (2) The pH dependence of catalytic activity for modified chymotrypsins is similar to that for native chymotrypsins (data not shown). (3) All modified samples were characterized using SDS–PAGE and no difference from the native form was observed (data not shown). This also indicates that intermolecular `sewing', which could be expected in the case of pyromellitic anhydride, does not take place. (4) The Km values of modified samples are the same order of magnitude as that for the native enzyme. The Km values of modified and native CT samples behave similarly: they increase with organic cosolvent concentration (data not shown).

We found that the values of the stabilization effect, expressed by organic cosolvent concentration at which enzyme activity returns to its initial value (C100), can be correlated with the increment of hydrophobicity ({Delta}{Delta}G) introduced by the modification (Figure 3Go). The values of ({Delta}{Delta}G) were calculated from Hansch hydrophobic increments of functional groups and atoms (Mozhaev et al., 1992Go; Leo et al., 1971Go) and are given in Table IGo. In the case of hydrophilic and hydrophobic modifications at small and moderate increments ({Delta}{Delta}G), the stabilization effect is proportional to the amounts of hydrophobicity introduced. It is interesting that left and right halves of the curve, corresponding to hydrophobization and hydrophilization of enzyme surface, are almost symmetric about the y-axis (Figure 3Go). At higher {Delta}{Delta}G the stabilization effect is almost independent of further increases in the increment.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Correlation of stabilizing effect of modification, expressed by DMF concentration (C100) up to which the activity of CT is at least 100% of its value in aqueous solution, with Hansch hydrophobic increments ({Delta}{Delta}G) of functional groups and atoms introduced by the modification.

 
In addition, we performed the modification of CT with a mixture of glyceric and pentylic aldehydes (glyc–pentCT) taken in equal amounts. The total degree of modification of this sample was determined as 11. From our experience, the reactivities with glyceric and pentylic aldehydes are identical, so one might suppose that the degrees of modification for them are almost equal and hence the {Delta}{Delta}G value should be about –40 kJ/mol (Table IIGo). Despite such a small increment, glyc–pentCT appeared to be the most stable of all modified samples studied in recent work – it was characterized by C100 = 43.5% in DMF.


View this table:
[in this window]
[in a new window]
 
Table II. The measured calorimetric parameters of denaturation in aqueous solution (20 mM MOPS, pH 7.5) for modified and non-modified chymotrypsin samples
 
To reveal the mechanisms by which the two types of modification affect the enzyme properties, we studied native and modified CT samples in water–organic mixtures using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC).

Circular dichroism

The samples analyzed by far-UV CD spectroscopy, glyc13CT (hydrophilic modification) and ibut7CT (hydrophobic modification), are fairly similar considering the catalytic activity in water–ethanol media (Figure 2Go). They are much more stable against inactivation than the native form. However, from the CD spectra we found that the structural properties of the analyzed samples in aqueous solution and in water–ethanol media are very different.

The CD spectrum for native CT (not shown) was similar to the published spectrum (Gorbunoff, 1971Go). For comparison, the values according to theoretical data are also presented. Upon addition of ethanol up to 20% (v/v) practically no changes in the CD spectra are observed. The values of secondary structure contents vary within the estimation error (Table IIIGo). The addition of more ethanol (30%, v/v) to the media gives rise to considerable changes in the structure: the ß-sheet content is increased mainly at the expense of {alpha}-helix and ß-turn. Very similar changes take place at higher ethanol concentrations (50 and 70%, v/v). The observed increase in ß-sheet content can be attributed to aggregation of the protein, which is known for CT under the given conditions (Kudryashova et al., 1997Go). It is worth noting that the decrease in catalytic activity is the sharpest between 25 and 30% (v/v) ethanol (Figure 2Go). Hence the inactivation of CT in water–ethanol mixtures can be accounted for by aggregation. The formation of ß-structures and loss of activity in 50% ethanol were reported for native CT also by another group (Sato et al., 2000Go).


View this table:
[in this window]
[in a new window]
 
Table III. The percentage of secondary structure elements, estimated from CD spectra using CDNN software (http://bioinformatik.biochemtech.unihalle.de/cd_spec/cdnn)
 
The CD spectrum of glyc13CT (Figure 4Go) in aqueous solution is less regular in comparison with native CT. Deconvolution of the CD spectrum indicates a lower percentage of ß-sheets and a higher percentage of {alpha}-helix and ß-turns (Table IIIGo). However, from the values of these variations one can judge that the secondary structure has not been distorted dramatically by the modification. Much more pronounced perturbations were observed upon addition of ethanol to the media (Figure 4Go). The corresponding values of secondary structure content change non-monotonically (Table IIIGo). At ethanol concentrations up to 30% (v/v) the {alpha}-helix content gradually decreases from 12.9 to 9.3%, probably leading to the slight increase of other structures. However, at higher ethanol concentrations the {alpha}-helix content increases about 6-fold, to 57.8% at 50% (v/v) EtOH. At the same time the ß-sheet content drops approximately 5-fold, from 30.2 to 6.2%. The random coil and ß-turn contents also decrease substantially. Instead of aggregation and complete loss of activity (which is the case for the native enzyme), for glyc13CT we observe the refolding to some new, `incorrect' structure, which is able to remain soluble and retains considerable catalytic activity (Figure 2Go). A phenomenon of such `incorrect' refolding to a structure with higher helix contents has been reported for some proteins on exposure to water–alcohol mixtures (Buck et al., 1993Go; Fan et al., 1993Go). Probably this is the way in which the protein can `adapt' to a non-natural environment.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. Circular dichroism spectra of glyc13CT in the far-UV region at various ethanol concentrations (v/v): 0, 0%; 1, 20%; 2, 30%; 3, 40%; 4, 45%; 5, 50%.

 
It was surprising to see that the far-UV CD spectrum of ibut7CT is extremely destructured (Figure 5Go) under conditions where the enzyme is fully active. Several computer programs were tried, but all of them failed to yield a reliable estimation of secondary structure contents in ibut7CT samples. However, we found that the CD spectrum is dramatically changed after inhibition of ibut7CT with PMSF. PMSF is an irreversible inhibitor of CT and a PMSF-inhibited sample can be considered as a model of an enzyme–substrate transition state. Figure 6Go shows the CD spectrum of PMSF-ibut7CT in comparison with ibut7CT. The molar ellipticity of the PMSF-inhibited sample is substantially higher and the corresponding percentage of secondary structure elements is similar to that of native CT (Table IIIGo). Apparently, the modification with butyric aldehyde led to partial reversible unfolding of the protein. However, it is possible that the core structure of the protein crucial for catalytic activity is still intact. Unlike native CT, the given `partially unfolded structure' can undergo considerable perturbations, but still remain soluble and retain its catalytic activity at high ethanol concentrations. On addition of PMSF the `partially unfolded structure' is reversed to a native-like form. Perhaps ibut7CT, as well as glyc13CT, is able to `adapt' to a non-natural medium. However, the mechanism of its `adaptation' is apparently different.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Circular dichroism spectra of ibut7CT in the far-UV region at various ethanol concentrations (v/v): 0, 0%; 1, 40%; 2, 50%; 3, 70%; 4, 80%.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6. Circular dichroism spectra in the far-UV region of ibut7CT (1) and ibut7CT inhibited with PMSF (2).

 
Differential scanning calorimetry

For DSC studies we selected glyceric aldehyde-modified CT with degree of modification 13 (hydrophilic modification, glyc13CT); and pentylic aldehyde-modified CT with degree of modification 11 (hydrophobic modification, pent11CT). As we mentioned, these samples were considerably more stable than native CT in respect of retention of catalytic activity in water–DMF mixtures (Figure 1Go). The parameters obtained by DSC indicate that in aqueous media both modifications lead to the formation of CT structure with higher conformational stability (Table IIGo).

We also followed the behavior of pent11CT and glyc13CT in water–DMF media using DSC techniques. For both modified and native CT, the addition of DMF causes an almost linear decrease in denaturation temperature and a non-linear decrease in enthalpy of denaturation. For native CT the excess heat capacity function contains a single peak, which apparently corresponds to a single cooperative system (Privalov, 1979Go). However, for both modified samples, particularly at 10–20% (v/v) DMF, the experimental excess heat capacity function shows the presence of several fractions or quasi-independent structural components (Privalov, 1981Go). We deconvoluted the experimental excess heat capacity functions as a superposition of several independent components and calculated the parameters for each of them (Figure 7Go).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7. The experimental excess heat capacity function of pent11CT in 20% (v/v) ethanol (as an example) can be deconvoluted as a superposition of two independent components. Solid line, experimental trace; dashed line, deconvolution.

 
From such a deconvolution, it can be judged that pent11CT is more likely to contain two components. The behavior of the less stable one (component 2) is similar to that of the native CT, whereas component 1 is considerably more stable. This can be judged from the changes in the corresponding {Delta}Hd and T1/2 with increase in DMF concentration (Figures 8 and 9GoGo). Perhaps the modification transforms some part of the protein globule into a stable domain, which can be resolved using DSC. It is also possible that the above components 1 and 2 represent two fractions of pent11CT: `successfully' and `unsuccessfully' modified molecules. The presence of residual activity at high concentrations of organic cosolvent can be accounted for by the functioning of a `successfully' modified domain or fraction.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8. Dependence of denaturation temperature (T1/2) on DMF concentration for pent11CT represented as a two-component system. {circ}, Native CT; {triangleup}, component 1 (less stable) of pent11CT; {square}, component 2 (more stable) of pent11CT.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 9. Dependence of denaturation enthalpy ({Delta}Hd) on DMF concentration for pent11CT represented as a two-component system. The total height of the column corresponds to the total entalpy of denaturation. Black, the contribution of component 1 (less stable); gray, the contribution of component 2 (more stable).

 
The experimental excess heat capacity function of glyc13CT is well fitted as a three-component system. Component 1 (the most stable) is obviously more stable in comparison with native CT, but components 2 and 3 are similar or even less stable (Figures 10 and 11GoGo). Component 1 could represent a `successfully' modified domain or fraction. Component 2 and especially component 3 are in fact `non-successfully' modified. Perhaps in the latter case, the modification of some `incorrect' residues took place. For example, owing to the higher degree of modification, amino groups crucial for maintaining the hydrophilic interaction network were modified. Also, the introduced hydrophilicity of glyceric aldehyde has not compensated for the loss of those interactions.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 10. Dependence of denaturation temperature (T1/2) on DMF concentration for glyc13CT represented as a three-component system. {circ}, Native CT; {blacksquare}, component 1 (the most stable) of pent11CT; {blacktriangleup}, component 2 of pent11CT; {blacklozenge}, component 3 (the least stable).

 


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 11. Dependence of denaturation enthalpy ({Delta}Hd) on DMF concentration for glyc13CT represented as a three-component system. The total height of the column corresponds to the total enthalpy of denaturation. Black, the contribution of component 3 (the least stable); white, the contribution of component 2; gray, the contribution of component 1 (the most stable).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The introduction of additional hydrophobicity, as well as hydrophilicity, on to the CT surface led to the formation of a more stable protein structure (domains or fractions), less vulnerable to denaturation in water–organic mixtures. It is most probable that the mechanisms of stabilization for two types of modifying agents are different. These conclusions can be drawn from the recent work. While the stabilization effect was not surprising to us in the case of hydrophilic modification (Khmelnitsky et al., 1991Go; Kudryashova et al., 1997Go), the mechanism of stabilization upon hydrophobic modification is unclear.

Replacements of surface-charged amino acids with hydrophobic ones using site-directed mutagenesis was reported to improve enzyme stability substantially (Arnold, 1988Go; Martinez and Arnold, 1991Go; Martinez et al., 1992Go; van den Burg et al., 1994Go). This approach works if the replaced hydrophilic residue is not crucial for maintaining salt bridges and a hydrogen-bond network. But how could such explanation be extended to the case of chemical modifications, where the sites of modification are not known?

From the chemical point of view, the reactivity of amino functions involved in multiple hydrophilic interactions will be lower. Hence they would be less susceptible to chemical modification. If that is so, uncompensated, unsatisfied surface residues (lysines and N-terminal amino acids) would have the highest reactivity. If our assumption is correct, all these residues are the first candidates for chemical modification. Substituted with a hydrophobic substance, they can be expected to contribute to protein stabilization in water–organic mixtures. However, if this mechanism was really working in our case, one could expect the stabilizing effect not only for pentylic and isobutyric aldehydes, but also for acetic aldehyde. Our data indicates that this is not so – no effect of the latter was observed. Therefore, to make the protein more stable it is not sufficient just to replace the unfavorable surface charge.

In our case, it could be supposed that additional hydrophobic groups introduced during modification create additional interactions with unfavorable hydrophobic regions on the protein surface. Apparently, this must contribute to protein stabilization (Lee and Richards, 1971Go; Chotia, 1984Go). The hydrophobic increment of acetic aldehyde is perhaps not sufficient to build such interactions.

Another possibility is that in the course of modification the distribution of modificator molecules around the protein globule is not even: more hydrophobic molecules would preferentially react with amino groups adjacent to uncompensated hydrophobic regions on the protein surface. If so, we have a kind of self-adjusting system, where the modifier `finds' the way to attach near the uncompensated region. Similar suggestions can be put forward for hydrophilic modification. For the examination of our hypothesis we performed the modification of CT with a mixture of glyceric and pentylic aldehydes (glyc–pentCT) taken in equal amounts. As mentioned in the Results section, the total degree of modification of this sample was determined as 11; also considering the reactivity with glyceric and pentylic aldehydes, the degrees of modification for them should be equal. Hence the {Delta}{Delta}G value should be about –40 kJ/mol (see Table IIGo). It is amazing that despite such a small increment, glyc–pentCT appeared to be the most stable of all modified samples studied in recent work. This is an indication that the most efficient stabilization is not achieved by hydrophilization or hydrophobization itself, but by `successful' modification. Perhaps in glyc–pentCT the structure has been optimally readjusted, so that both stabilization mechanisms are working.


    Notes
 
2 To whom correspondence should be addressed. E-mail: alvin{at}ineos.ac.ru. Back


    Acknowledgments
 
We thank Dr Vadim V.Mozhaev of EnzyMed, Iowa City, IA, USA for his substantial contribution to this work in the initial stages. We also thank Martina Reddington of Ireland for checking our English. This work was supported by the Russian program `The New Methods in Bioengineering: Engineering Enzymology'.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arnold,F.H. (1988) Protein Eng., 2, 21–25.[Abstract]

Arnold,F.H. and Volkov,A.A. (1999) Curr. Opin. Chem. Biol., 3, 54–59.[ISI][Medline]

Buck,M., Radford,S.E. and Dobson,C.M. (1993) Biochemistry, 32, 669–678.[ISI][Medline]

Chotia,C. (1984) Annu. Rev. Biochem., 53, 537–572.[ISI][Medline]

De Marco,A., Lecomte,J.T.J. and Llinas,M. (1981) Eur. J. Biochem., 119, 483–490.[ISI][Medline]

Fahrney,D. and Gold,A.M. (1963) J. Am. Chem. Soc., 85, 997–1000.[ISI]

Fan,P., Bracken,C. and Baum,J. (1993) Biochemistry, 32, 1573–1582.[ISI][Medline]

Fields,R. (1971) Biochem. J., 124, 581–590.[ISI][Medline]

Gladilin,A.K., Kudryashova,E.V., Vakurov,A.V., Izumrudov,V.A., Mozhaev,V.V. and Levashov,A.V. (1995) Biotechnol. Lett., 17, 1329–1334.[ISI]

Gorbunoff,M. (1971) Biochemistry, 10, 250–257.[ISI][Medline]

Grinberg,V.Ya., Burova,T.V., Haertle,T. and Tolstoguzov,V.B. (2000) J. Biotechnol., 79, 269–280.[ISI][Medline]

Halling,P.J. (2000) Curr. Opin. Chem. Biol., 4, 74–80.[ISI][Medline]

Khmelnitsky,Y.L. and Rich,J.O. (1999) Curr. Opin. Chem. Biol., 3, 47–53.[ISI][Medline]

Khmelnitsky, Yu.L., Belova,A.B., Levashov,A.V. and Mozhaev,V.V. (1991) FEBS Lett., 284, 267–269.[ISI][Medline]

Klibanov,A.M. (2001) Nature, 409, 241–246.[ISI][Medline]

Kudryashova,E.V., Belova,A.B., Vinogradov,A.A. and Mozhaev,V.V. (1994) Russ. J. Bioorg. Chem., 20, 274–280.

Kudryashova,E.V., Gladilin,A.K., Vakurov,A.V., Heitz,F., Levashov,A.V. and Mozhaev,V.V. (1997) Biotechnol. Bioeng., 55, 267–277.[ISI]

Laemmli,U.K. (1970) Nature, 227, 680–684.[ISI][Medline]

Lee,B. and Richards,F.M. (1971) J. Mol. Biol., 55, 187–211.

Leo,A., Hansch,C. and Elkins,D. (1971) Chem. Rev., 71, 525–643.[ISI]

Martinez,P. and Arnold,F.H. (1991) J. Am. Chem. Soc., 113, 6336–6337.[ISI]

Martinez,P., van Dam,M.E., Robinson,A.C., Chen,K. and Arnold,F.H. (1992) Biotechnol. Bioeng., 39, 141–147.[ISI]

Maurel,P. (1978) J. Biol. Chem., 253, 1677–1683.[ISI][Medline]

Mozhaev,V.V., Silksnis,V.A., Melik-Nubarov,N.S., Galkantaite,N.Z., Denis,G.J., Butkus,E.P., Zaslavsky,B.Yu., Mestechkina,N.M. and Martinek K. (1988) Eur. J. Biochem., 173, 147–154.[Abstract]

Mozhaev,V.V., Khmelnitsky,Y.L., Sergeeva,M.V., Belova,A.B., Klyachko,N.L., Levashov,A.V. and Martinek,K. (1989) Eur. J. Biochem., 184, 597–602.[Abstract]

Mozhaev,V.V., Melik-Nubarov,N.S., Siksnis,V.A., Levitsky,V.Y. and Martinek,K. (1992) Biotechnol. Bioeng., 40, 650–662.[ISI]

Mozhaev,V.V., Kudryashova,E.V., Efremova,N.V. and Topchieva,I.N. (1996) Biotechnol. Tech., 10, 849–854.[ISI]

Oldfield,C. (1994) Biotechnol. Genet. Eng. Rev., 12, 255–327.[Medline]

Privalov,P.L. (1979) Adv. Protein Chem., 33, 167–241.[Medline]

Privalov,P.L. (1981) Adv. Protein Chem., 35, 1–104.

Privalov,P.L. and Potekhin,S.A. (1986) Methods Enzymol., 131, 4–51.[Medline]

Sato,M., Sasaki,T., Kobayashi,M. and Kise,H. (2000) Biosci. Biotechnol. Biochem., 64, 2552–2558.[ISI][Medline]

Schonbaum,G.R., Zerner,B. and Bender,M. (1961) J. Biol. Chem., 236, 2930–2938.[ISI][Medline]

Teeter,M.M., Mazer,J.A. and L'Italien,J.J. (1981) Biochemistry, 20, 5437–5443.[ISI][Medline]

Tuena de Gomez-Puyou,M. and Gomez-Puyou,A. (1998) Crit. Rev. Biochem. Mol. Biol., 33, 53–89.[Abstract]

van den Burg,B., Dijkstra,B.W., Vriend,G., van der Vinne,B., Venema,G. and Eijsink,V.G. (1994) Eur. J. Biochem., 220, 981–985.[Abstract]

Volini,M. and Tobias,P. (1969) J. Biol. Chem., 244, 5105–5109.[Abstract/Free Full Text]

West,J.B., Hennen,W.J., Lalonde,J.L., Bibbs,J.A., Zhong,Z., Meyer,E.F.,Jr. and Wong,C.H. (1990) J. Am. Chem. Soc., 112, 5313–5320.[ISI]

Zaks,A. and Klibanov,A.M. (1984) Science, 224, 1249–1251.[ISI][Medline]

Zaks,A. and Klibanov,A.M. (1985) Proc. Natl Acad. Sci. USA, 82, 3192–3196.[Abstract]

Received February 16, 2001; revised June 8, 2001; accepted June 18, 2001.