Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila.

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

Novel multienzyme oxidative biocatalyst for lignin bioprocessing.

A novel multienzyme biocatalyst, based on coimmobilization of the laccase and horseradish peroxidase by cross linking and layer-by-layer coating with polyelectrolyte, was designed, synthesized and applied at the development of an oxidative cascade process on lignin. The efficiency and specificity of the new LbL-multienzyme system, the occurrence of a synergy of the co-immobilized enzymes, the lignin oxidation pathway and the nature of the structural modifications occurred in treated lignins have been investigated in the present effort by means of GPC analysis and quantitative (31)P NMR techniques.

Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase.

Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni(2+) for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, beta and charge transfer (CT1) bands absorption, shift in the Soret, beta and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the alpha-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni(2+) were studied by following the H(2)O(2)-mediated oxidation of o-dianisidine and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni(2+) concentrations (20-50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni(2+). Prolonged incubation would induce complete inhibition at lower Ni(2+) concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni(2+) altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni(2+) concentrations. HRPC was found to have four potential attachment sites for Ni(2+) which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.

Increased thermal and organic solvent tolerance of modified horseradish peroxidase.

Horseradish peroxidase (HRP) was modified by maleic anhydride and citraconic anhydride. The thermal and organic solvent tolerances of native and modified enzyme were compared. These chemical modifications of HRP increased their thermostability both in aqueous buffer and some organic solvents, and also enhanced their tolerances of some organic solvents. We have studied the unfolding of native and modified HRP by heat to determine the conformational stability. The temperature at the midpoint of thermal denaturation (T(m)) was increased upon modification. Both enthalpy change (DeltaH(m)) and entropy change (DeltaS(m)) for unfolding of modified enzyme at T(m) were decreased compared with native enzyme. Circular dichroism studies proved that these modifications changed the conformation of HRP. The improvements of stability are related to side chain reorientations of aromatics upon both modifications.

Hofmeister effects in biology: effect of choline addition on the salt-induced super activity of horseradish peroxidase and its implication for salt resistance of plants.

The effect of choline addition on the salt-induced super activity of horseradish peroxidase (HRP) is investigated. HRP is presented in the literature as an efficient H(2)O(2) scavenger, and choline is the precursor of glycine betaine, a strong osmoprotectant molecule. Both the regulations of H(2)O(2) and of osmoprotectant concentrations are implicated in plants in order to counteract salt-induced cell damage. For the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), sulfate anions were found to play a crucial role in the increase of HRP activity. This induced super activity can be strongly reduced by adding choline chloride. The phenomena provide an example of physicochemical Hofmeister effects playing a central regulatory role in an important biological system.

Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase.

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656-666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 28.7 mm Hg(-1) s(-1) in the presence of BHA. The quenching rate of BHA is 4000 s(-1). The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O(2) diffusion and flexibility in heme proteins.

Phospholipid assisted folding of a denatured heme protein: effect of phosphatidylethanolamine.

The role of the aminophospholipid, phosphatidylethanolamine (PE), has been well established to act as a non-protein molecular chaperone in the folding and assembly of polytopic membrane proteins. However, such studies with soluble proteins have not been done so far and in particular with the heme proteins. We have used the heme enzyme, horseradish peroxidase (HRP), as the model heme protein and studied the effect of different phospholipids on its refolding from denatured state. Dimyristoylphosphatidylethanolamine (DMPE), a bilayer-forming PE, was able to increase the reactivation yield of denatured HRP upon 30min refolding at 25 degrees C. However, dioleoylphosphatidylethanolamine (DOPE), containing one double bond in the fatty acid chains, which does not favour bilayer organization, did not support proper refolding. The phospholipids with N-methylated head groups, phosphatidylcholines, e.g., DMPC and DOPC showed differential effects when DMPC remained mostly non-supportive while DOPC on the contrary led to inhibition of the refolding of the denatured heme enzyme. Fluorescence spectroscopic studies also indicated changes in the microenvironments of the heme moiety and the single tryptophan residue of HRP in presence of the aminophospholipid.

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity.

Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.

Factors regulating the effect of IL-4 on intestinal epithelial barrier function.

BACKGROUND: Inflammatory bowel disease is associated with an imbalance in cytokine production and defective intestinal barrier function. Previous studies indicate that IL-4, a cytokine increased in food allergy and in early Crohn's disease, enhances epithelial permeability. Here, we characterized the mechanism of action of IL-4 on cultured epithelial cells and examined if the anti-inflammatory cytokines, TGF-beta or IL-10, can modulate the effects of IL-4. METHODS: Confluent monolayers of human T84 epithelial cells were cultured with IL-4 alone or in combination with IL-10 or TGF-beta or with inhibitors of protein synthesis and blockers of IL-4 receptor signalling pathways. Permeability was evaluated by measuring transepithelial resistance (TER), flux of (3)H-fMLP (a small bacterial tripeptide) and horseradish peroxidase (HRP) (a macromolecule). RESULTS: T84 cells cultured with IL-4 showed a significant drop in TER as well as an increased flux of (3)H-fMLP and HRP. Co-treatment with IL-10 did not improve TER, whereas TGF-beta attenuated the resistance drop. However, neither TGF-beta nor IL-10 were able to correct the increased (3)H-fMLP flux. In contrast, the increased HRP flux caused by IL-4 was inhibited by both IL-10 and TGF-beta. TGF-beta and IL-10 significantly reduced IL-4-enhanced values for endosomal area and paracellular spaces containing HRP. Inhibitor studies indicated the requirement for protein synthesis and the involvement of phosphatidylinositol 3-kinase. CONCLUSIONS: These results provide new insights into the regulation of intestinal barrier function and may suggest a novel approach in the treatment of intestinal inflammation.

Transendothelial transport of renin-angiotensin system components.

BACKGROUND: Vascular (interstitial) angiotensin (ANG) II production depends on circulating renin-angiotensin system (RAS) components. Mannose 6-phosphate (man-6-P) receptors and angiotensin II type 1 (AT(1)) receptors, via binding and internalization of (pro)renin and ANG II, respectively, could contribute to the transportation of these components across the endothelium. OBJECTIVE: To investigate the mechanism(s) contributing to transendothelial RAS component transport. METHODS: Human umbilical vein endothelial cells were cultured on transwell polycarbonate filters, and incubated with RAS components in the absence or presence of man-6-P, eprosartan or PD123319, to block man-6-P, AT(1) and angiotensin II type 2 (AT(2)) receptors, respectively. RESULTS: Apically applied (pro)renin and angiotensinogen slowly entered the basolateral compartment, in a similar manner as horseradish peroxidase, a molecule of comparable size that reaches the interstitium via diffusion only. Prorenin transport was unaffected by man-6-P. Apical ANG I and ANG II rapidly reached the basolateral fluid independent of AT(1) and AT(2) receptors. Basolateral ANG II during apical ANG I application was as high as apical ANG II, whereas during apical ANG II application it was lower. During basolateral ANG I application, ANG II generation occurred basolaterally only, in an angiotensin-converting enzyme (ACE)-dependent manner. CONCLUSIONS: Circulating (pro)renin, angiotensinogen, ANG I and ANG II enter the interstitium via diffusion, and interstitial ANG II generation is mediated, at least in part, by basolaterally located endothelial ACE.

Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents.

Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase.

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.

Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.

Ionic strength and pH effect on the Fe(III)-imidazolate bond in the heme pocket of horseradish peroxidase: an EPR and UV-visible combined approach.

The effects of chloride, dihydrogenphosphate and ionic strength on the spectroscopic properties of horseradish peroxidase in aqueous solution at pH=3.0 were investigated. A red-shift (lambda=408 nm) of the Soret band was observed in the presence of 40 mM chloride; 500 mM dihydrogenphosphate or chloride brought about a blue shift of the same band (lambda=370 nm). The EPR spectrum of the native enzyme at pH 3.0 was characterized by the presence of two additional absorption bands in the region around g=6, with respect to pH 6.5. Chloride addition resulted in the loss of these features and in a lower rhombicity of the signal. A unique EPR band at g=6.0 was obtained as a result of the interaction between HRP and dihydrogenphosphate, both in the absence and presence of 40 mM Cl-. We suggest that a synergistic effect of low pH, Cl- and ionic strength is responsible for dramatic modifications of the enzyme conformation consistent with the Fe(II)-His170 bond cleavage. Dihydrogenphosphate as well as high chloride concentrations are shown to display an unspecific effect, related to ionic strength. A mechanistic explanation for the acid transition of HRP, previously observed by Smulevich et al. [Biochemistry 36 (1997) 640] and interpreted as a pure pH effect, is proposed.

Structural changes of horseradish peroxidase in presence of low concentrations of urea.

The presence of very low concentrations of the widely used denaturant urea induces structural changes in the monomeric heme-containing enzyme, horseradish peroxidase (HRP). Structural alterations in the protein were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of urea, even in concentrations below 100 mm, were higher than those measured in absence of the denaturant. The fluorescence emission maximum of 1, 8-ANS, used as a probe for monitoring conformational changes in the enzyme, was blue-shifted from 530 nm in aqueous buffer to 518 nm when incorporated in native HRP. This blue shift increased further by 3 nm in presence of HRP preincubated with 100 mm urea, whereupon it steadily decreased with increasing urea concentration to become zero at 8 m urea. The mean fluorescence lifetime of 1,8-ANS incorporated in HRP was much higher than that of ANS in aqueous buffer, and showed continuous variation with the concentration of urea in which the enzyme was incubated. Systematic changes in the microenvironment of the heme moiety in HRP were also reflected in the visible CD spectra of the enzyme incubated with low concentrations of urea. These results are consistent with those of our earlier studies performed with the denaturant guanidinium chloride and indicate structural relaxation of HRP, with retention of enzymatic activity and native-like secondary structure, in presence of millimolar concentrations of urea.

Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase.

The influence of N-linked glycans on the stability of glycoproteins has been studied using horseradish peroxidase isoenzyme C (HRP), which contains eight asparagine-linked glycans. HRP was deglycosylated (d-HRP) with trifluoromethanesulfonic acid and purified to an enzymatically active homogeneous protein containing (GlcNAc)2 glycans. The thermal stability of HRP and d-HRP at pH 6.0, measured by residual activity, was indistinguishable and showed transition midpoints at 57 degrees C, whereas the unfolding in guanidinium chloride at pH 7.0, 23 degrees C was 2-3-fold faster for d-HRP than for HRP. The results are compatible with a glycan-induced decrease in the dynamic fluctuation of the polypeptide chain.

Effects of cyclosporin A on paracellular and transcellular transport of horseradish peroxidase in perfused rat livers.

The immunosuppressive agent cyclosporin A (CsA) is known to cause cholestasis. Transcellular and paracellular transport of macromolecules contribute, albeit to a minor extent, to bile formation, but little is known about the effects of CsA on these pathways. The aims of this study were to investigate the influence of CsA on tight junction (paracellular) permeability and on transcytotic vesicular pathways labeled with horseradish peroxidase (HRP) in perfused rat liver. Livers from male Sprague-Dawley rats were perfused with Krebs-Henseleit buffer (albumin 1%, RBC 20%, and amino acid mixture). Taurodehydrocholate (1 microM/min) was coinfused into the portal vein; 1 microg/ml of CsA, dissolved in Cremophor-EL (CsA livers), or the vehicle alone (CEL livers), was added to the medium. Tight junction permeability was assessed by administering HRP (25 mg) as a short pulse to perfused rat livers, operating under single-pass conditions. Under such conditions, HRP output into the bile shows two components: an initial peak at approximately 3-5 min, corresponding to paracellular transfer across tight junctions, and a second peak at approximately 15 min, corresponding to vesicular transport. Furthermore, we assessed the vesicular transport pathway by examining HRP-labeled vesicles in the perisinusoidal (PS) and pericanalicular (PC) areas using ultrastructural morphometric analysis. To analyze HRP in hepatocytes and to study rapid and late transcytotic vesicular pathways, a 1-min pulse of a high dose of HRP (500 and 200 mg, respectively) was given. Two and 18 min after single-pass perfusion the livers were fixed with 2.5% glutaraldehyde-0.8% paraformaldehyde in 0.1 mM cacodylate buffer, pH 7.8. The total pericanalicular area, the HRP-containing structures, were quantified morphometrically in liver samples. At concentrations of 1.2 microg/ml, CsA produced a twofold increase in the paracellular transfer of HRP to bile. The areas under the second peak (transcellular vesicular pathway) of the biliary HRP secretion curve were similar in CEL- and CsA-treated livers. Morphometric analysis confirmed that CsA treatment did not affect the percentage area of HRP-labeled vesicles in either the pericanalicular or in the perisinusoidal area at 2 min (rapid pathway) and 18 min (late pathway). These results indicate that CsA increases tight junctional permeability whereas it does not inhibit rapid or late transcytotic vesicle pathways.

Importance of glycolipid synthesis for butyric acid-induced sensitization to shiga toxin and intracellular sorting of toxin in A431 cells.

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitization (> 1000-fold) is accompanied by an increase in the fraction of cell-associated toxin transported to the Golgi apparatus and to the endoplasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have not investigated the importance of this change by testing whether glycolipid synthesis is required for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inhibition of N-acyltransferase with fumonisin B1, by inhibition of glucosylceramide synthetase by PDMP or PPMP, or by inhibition of serine palmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid-induced change in sensitivity and the increase in the fraction of cell-associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoroalanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length of glycosphingolipids is important for intracellular sorting and translocation of Shiga toxin to the cytosol.

Protein stabilisation using additives based on multiple electrostatic interactions.

A method of elevating the storage lifetime of purified proteins has been discovered which appears to confer stability to all proteins investigated and may therefore be classed as generic in action. The basic methodology involves the formation of multiple electrostatic complexes between the protein and selected soluble polyelectrolytes to give protein-polyelectrolyte (PP) complexes and then to add solutions of polyalcohols or other compounds containing multiple hydroxyl groups. Dehydration of the resulting solution by vacuum evaporation, freeze drying or forced air convection produces a dry film or powder of stabilised protein. The method has been used mainly in the preparation of active enzymes for analytical tests. It has also been found that the formation of PP complexes also enhances the stability of enzymes in solution and the technique may be applicable to the stabilisation of virus suspensions by polycations. Examples of stabilised enzymes prepared by these methods are given and the proposed mechanism of stabilisation and applicability of the method to shelf-stable vaccine products are discussed.