Prevention of thermal inactivation and aggregation of lysozyme by polyamines Motonori Kudou 1 , Kentaro Shiraki 1 , Shinsuke Fujiwara 2 , Tadayuki Imanaka 3 and Masahiro Takagi 1 1 School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan; 2 Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan; 3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan Proteins tend to form inactive aggregates at high tempera- tures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 m M ), more than 30% of 0.2 mgÆmL )1 lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggre- gates by heat treatment (98 °C for 30 min). 1 However, in the presence of 50 m M spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 m M arginine and guanidine, while it was maintained at  50% in the presence of 100 m M spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins. Keywords: protein misfolding; protein aggregation; poly- amine; thermal inactivation. Proteins fold into their unique native structure, even in vitro. However, they tend to form undesirable and uncontrollable aggregates during the unfolding and refolding processes, both in the laboratory and even in their natural environ- ment in living cells. Protein aggregation is a major problem in the large-scale production of recombinant proteins [1–3], as well as in living cells, where it may lead to the occurrence of fatal diseases 2 [4,5]. Various techniques have been developed to prevent the formation of protein aggregates. One of the major approaches used to prevent protein aggregation is the addition of small molecules to the solution. This is a relatively simple method compared with using chaperon systems [6–8]. The small molecular additives used to prevent the formation of protein aggregates are classified as protein- denaturing reagents or others. Denaturants, typically guanidine and urea, weaken the hydrophobic intermole- cular interaction of proteins [9,10]. Detergents, such as Triton-X100 and SDS, are stronger protein-denaturing reagents than denaturants [10,11]. Not only do these reagents dissolve aggregates and inclusion bodies but they also unfold the native structure of proteins. Accordingly, the concentration at which this type of reagent is effective at preventing the aggregation and inactivation of proteins is hard to determine. 3 Arginine (Arg) is a nondenaturing reagent that has been used widely as an additive to prevent protein aggregation [9–12]. Arg does not destabilize the native structure, having only a minor effect on protein stability [11,13], and enhances the solubility of aggregate-prone molecules. Because of its beneficial properties, Arg has been used for various proteins and situations. However, the effect of Arg and other nondenaturing additives does not completely solve the aggregation problem. The development of better additives for preventing protein aggregation has been long awaited. In this article, we focus on naturally occurring poly- amines [putrescine, NH 2 (CH 2 ) 4 NH 2 ; spermidine, NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 ;spermine,NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH(CH 2 ) 3 NH 2 ] as small molecular additive candidates for preventing heat-induced aggregation and inactivation of proteins. There are a large number of different polyamines 4 in hyperthermophiles [14–16], which suggests that poly- amines have a biophysical role in the adaptation of hyperthermophilic proteins to high temperature environ- ments. Although it has been reported that polyamines bind to biomolecules (DNA, RNA, and platelets) by electrostatic interactions [17–19], at present no research has been published regarding the role of polyamines on thermal aggregation and inactivation of proteins. Materials and methods Materials Hen egg white lysozyme and betaine/HCl were purchased from Sigma Chemical Co. All amino acids, guanidine/HCl, urea, putrescine/2HCl, spermidine/3HCl, and spermine/ 4HCl were purchased from Wako Pure Chemical Industries (Osaka, Japan). Micrococcus lysodeikticus for the kinetics Correspondence to K. Shiraki, School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan. Fax: + 81 761 51 1655, Tel.: + 81 761 51 1657, E-mail: kshiraki@jaist.ac.jp Abbreviations: DCp, heat capacity change; DH, enthalpy change; DSC, differential scanning calorimetry; T m , midpoint temperature of thermal unfolding. Enzymes: lysozyme (EC 3.2.1.17). (Received 29 July 2003, revised 27 August 2003, accepted 23 September 2003) Eur. J. Biochem. 270, 4547–4554 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03850.x assay of lysozyme was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Trimethylamine-N-oxide was purchased from Aldrich Chemical Company, Inc. All other chemicals used were of high-quality analytical grade. Heat-induced aggregation of lysozyme Heat-induced aggregates of lysozyme were quantified as follows. 5 Solutions, containing 0.2 mgÆmL )1 lysozyme in 50 m M sodium-phosphate buffer (pH 6.5) and different concentrations of additives, were prepared. All stock solutions for additives and protein were dissolved in 50 m M sodium-phosphate buffer (pH 6.5) 6 and adjusted to pH 6.5 by NaOH or HCl before sample preparation. After heat treatment at 98 °C, the samples were centrifuged at 15 000 g for 20 min. The absorbance of the supernatants was monitored by using a Jasco spectrophotometer model V-550 (Japan Spectroscopic Company, Tokyo, Japan) to determine the concentration of soluble lysozyme, using an extinction coefficient of 2.63 cm )1 per mgÆmL )1 7 [20]. Residual activity of lysozyme after heat treatment The bacteriolytic activity of lysozyme was estimated as follows [21]. A 1.5 mL volume of 0.5 mgÆmL )1 M. lys- odeikticus solution prepared in 50 m M sodium-phosphate buffer (pH 6.5) was mixed with 20 lL of the heat-treated samples containing 0.2 mgÆmL )1 lysozyme and 100 m M additive. The decrease in light scattering intensity of the solution was monitored by measuring the absorbance (A)at 600 nm. The rate constant of inactivation was determined by fitting the data to a linear extrapolation. CD spectra Far-ultraviolet CD spectra were measured using a Jasco spectropolarimeter, model J-720 W, equipped with a thermal incubation system. The far-ultraviolet CD spectrum of lysozyme was measured at a protein concentration of 0.1 mgÆmL )1 , in a 2-mm cuvette. Differential scanning calorimetry Differential scanning calorimetry (DSC) for a mixture of lysozyme and an additive was performed using a nano- DSCII Differential Scanning Calorimeter 6100 (Calori- metry Sciences Corporation, UT, USA) with a cell volume of 0.299 mL and at a scanning rate of 2 °CÆmin )1 . Degassing during the calorimetric experiment was prevented by maintaining an additional constant pressure of 2.5 bars over the liquid in the cell. The samples were 4.0 mgÆmL )1 lysozyme in 50 m M sodium- phosphate buffer (pH 6.5) or sodium-acetate buffer (pH 4.4) together with 100 m M additive. Solutions con- taining additives were dialysed to adjust their pH to 6.5 or 4.4. The enthalpy change (DH), heat capacity change (DC p ), and midpoint temperature of thermal unfolding (T m ) were determined by a conventional method, as described previously [22]. Results Heat-induced aggregation of lysozyme Hen egg white lysozyme (pI ¼ 11) was used as a model protein because its mechanism of refolding and misfolding has been extensively studied [12,13,23–27]. Lysozyme can preferentially refold into its native structure from thermally unfolded states, while under neutral pH it tends to form irreversible aggregates during heating [23–25]. The amount of heat-induced aggregates produced from 0.2 mgÆmL )1 lysozyme,whenheatedto98°C for 30 min, was measured in the presence of various additives at pH 6.5 (Fig. 1). The amount of aggregates gradually Fig. 1. The amount of heat-induced aggregates produced in the presence of various additives. Solutions containing 0.2 mgÆmL )1 lysozyme (pH 6.5) and various concentrations of additives were heated at 98 °C for 30 min. After heat treatment, the amount of aggregates was calculated by determining the soluble concentration of lysozyme by centrifugation. (A) Arginine (Arg), (d); glycine (Gly), (s); guanidine, (h). (B) Betaine, (d); trimethylamine-N-oxide, (s); putrescine, (m); spermidine, (h); spermine, (j). (C) NaCl, (d); KCl, (s); urea, (j). 4548 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 decreased with increasing concentrations of Arg or guanidine from 0 to 0.5 M (Fig. 1A). In contrast,  50 m M spermidine and spermine completely prevented the thermal aggregation of lysozyme (Fig. 1B). The aggregation curve for putrescine, the smallest polyamine used in this study, 8 was essentially identical to those of Arg and guanidine. On the other hand, small ammonium ions (trimethylamine-N-oxide and betaine) did not prevent heat-induced aggregation (Fig. 1B). Other additives, such as glycine (Gly), urea, NaCl, or KCl did not prevent heat- induced aggregation (Fig. 1A,C). These data indicate that spermidine and spermine prevent heat-induced aggrega- tion of lysozyme better than the other additives tested in this study. Aggregation of lysozyme as a function of heating time and protein concentration Figure 2A shows the time course of heat-induced aggrega- tion with 100 m M of each additive. After 4 min, the amount of deposited aggregates showed an increase in the absence of any additives. However, in the presence of Arg, the amount of aggregates gradually increased after 10 min. In contrast 9 , with spermine, no aggregates were observed, even after heat treatmentat98°C for 40 min. Figure 2B shows the dependence of aggregation upon the initial concentration of lysozyme. In the absence of any additives, the concentration of soluble proteins reached a plateau at  0.07 mgÆmL )1 . Further increase of the protein concentration resulted in a gradual increase in the soluble concentration of lysozyme, from 0.07 mgÆmL )1 to 0.14 mgÆmL )1 . In the presence of 100 m M spermidine or spermine, no aggregates were observed at a protein concentration of < 0.4 mgÆmL )1 . With an increasing con- centration of lysozyme, the curve reached a plateau at  0.7 mgÆmL )1 . Interestingly, putrescine was clearly less effective than spermidine, whereas spermine was as effective as spermidine. This implies that an important factor required for polyamines to prevent protein aggregation is the presence of a secondary amine, rather than the number of cations or molecular mass. Aggregation by cooling We examined the heat-induced aggregation of lysozyme during cooling (Fig. 3A). Protein solutions of 0.2 mgÆmL )1 lysozyme, prepared in 50 m M sodium-phosphate buffer (pH 6.5) containing 100 m M additive, were heated at 98 °C for 30 min and then cooled from 98 °Cto50°Cbyusinga thermal controller. The concentration of soluble protein slightly decreased with cooling time. The slightly negative correlation between cooling time and concentration of soluble protein may be explained by the prolonged therm- ally unfolded state of the protein with longer cooling times. At temperatures above 84 °C, lysozyme was fully unfolded by heating (Fig. 3B). These data indicate that lysozyme aggregated during the heat treatment, rather than during the cooling. Fig. 2. Heat-induced aggregation of lysozyme was dependent on the incubation time and protein concentration. (A) The solutions containing 0.2 mgÆmL )1 lysozyme (pH 6.5) and 100 m M arginine (Arg) (s), spermine (j), or no additive (d), were heated at 98 °C. After heat treatment, the amount of aggregates was calculated by determining the soluble concentration of lysozyme by centrifugation. (B) The horizontal axis shows the concentration of lysozyme in samples containing different concentrations of lysozyme and 100 m M additive at pH 6.5. After heat treatment at 98 °C for 30 min, the soluble concentrations of lysozyme were determined and plotted on the vertical axis. No additive, (d); Arg, (s); putrescine, (m); spermidine, (h); or spermine (j). Ó FEBS 2003 Prevention of thermal aggregation by polyamines (Eur. J. Biochem. 270) 4549 Heat inactivation of lysozyme The recovery of enzymatic activity after heat treatment is another criterion used to estimate the effect of additives because it is the most reliable measure of whether additives prevent irreversible misfolding as well as aggregation. Figure 4 shows the residual activity of lysozyme after heat treatment at 98 °C. In the absence of additives, the inactivation curves of 0.2 and 1.0 mgÆmL )1 lysozyme fitted well to single-exponential equations (Fig. 4A). The inactivation rate constants for 0.2 and 1.0 mgÆmL )1 lyso- zyme were 0.067 and 0.21 min )1 , respectively. The heated samples, containing 1.0 mgÆmL )1 lysozyme (black circles in Fig. 4A), were resolved by the addition of guanidine/HCl (to a final concentration of 4.0 M ) 10 and vortexing for 15 min. These samples were diluted 10-fold by 50 m M sodium- phosphate buffer (pH 6.5), after which the residual activities of the samples were measured (Fig. 4A). However, the inactivation rate constant of the resolved sample was 0.23 min )1 , which was almost identical to that of the Fig. 3. Heat-induced aggregation is influenced by cooling time. (A) After heat treatment at 98 °C for 30 min, the protein solutions were cooled from 98 °Cto50°C for respective periods of time, and then the concentrations of soluble lysozyme were determined. The solutions were 0.2 mgÆmL )1 lysozyme and 50 m M sodium phosphate buffer (pH 6.5). The line shows the least-square fit for the raw data. (B) Far-ultraviolet CD spectra of lysozyme in 50 m M sodium-phosphate buffer (pH 6.5) at different temperatures. Fig. 4. Effect of additives on the time course of heat inactivation. Samples containing 0.2 mgÆmL )1 (open symbols) or 1.0 mgÆmL )1 (closed symbols) lysozyme, in 50 m M sodium phosphate buffer (pH 6.5) containing 100 m M additive, were heated at 98 °C. (A) Circles, no additives; crosses, samples of the closed circles resolved by 4.0 M guanidine/HCl and refolded by dilution. (B) Circles, putrescine; squares, spermidine. (C) Circles, spermine; squares, arginine (Arg). The continuous curves show least-square fitting of the respective data with single-exponential equations. 4550 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 unresolved sample. These facts indicate that the inactivated molecules, under the experimental conditions used in this study 11 , were mainly stabilized by covalent bonds (probably disulfide exchanges) rather than by noncovalent inter- actions. In the presence of 100 m M putrescine, the inactivation curves of 0.2 and 1.0 mgÆmL )1 lysozyme depended on the protein concentration, as shown by single-exponential equations 12 (Fig. 4B). The inactivation rate constants for 0.2 and 1.0 mgÆmL )1 lysozyme with 100 m M putrescine were 0.023 and 0.11 min )1 , respectively, which were two- to threefold slower than those in the absence of additives. Interestingly, in the presence of 100 m M spermidine and spermine, the inactivation curve of 1.0 mgÆmL )1 lysozyme was identical to that of 0.2 mgÆmL )1 lysozyme. The rate constants of inactivation in the presence of spermidine and spermine were 0.020 and 0.034 min )1 , respectively (Fig. 4B,C); these constants were one order of magnitude slower than those of 1.0 mgÆmL )1 lysozyme in the absence of additives. In the presence of 100 m M Arg, the inactivation rate constants for 0.2 and 1.0 mgÆmL )1 lysozyme were 0.045 and 0.19 min )1 , respectively (Fig. 4C). Arg also prevented the heat inactivation of lysozyme; however, the preventive effect was clearly lower than that induced by spermidine or spermine. In order to elucidate the versatility of polyamines, various additives were tested by measuring the residual activities (Table 1). After heat treatment for 30 min, total residual activities of 52% and 57%, for 0.2 mgÆmL )1 lysozyme, were recovered by the addition of 100 m M spermidine and spermine, respectively. On the other hand, the residual activity was < 5%, for most of the other additives (even 100 m M Arg), 13 which is an order of magnitude lower than for spermidine and spermine. Also, at a protein concentra- tion of 1.0 mgÆmL )1 and heat treatment for 10 min, polyamines prevented heat inactivation of lysozyme more effectively than the other additives (Table 1). DSC analysis To reveal the effect of additives on protein stability, thermodynamic parameters were determined using DSC. Representative DSC curves of lysozyme in the presence of 100 m M additive are shown in Fig. 5, and the thermo- dynamic parameters derived from nonlinear least-squares fit of the DSC data are listed in Table 2. DSC curves showed full reversibility at pH 4.4, but not at pH 6.5, so enthalpy change (DH) and heat capacity changes (DC p ) are listed only at pH 4.4. At pH 6.5, the T m of lysozyme was 77.3 °C in the absence of additives. The addition of 100 m M polyamines and Arg slightly increased the T m of lysozyme by  1 °Cand0.5°C, respectively, whereas addition of other additives did not alter the values compared with no additive 14 .Theincreased T m values at neutral pH are responsible for preventing irreversible aggregation during DSC measurement. At pH 4.4, the presence of polyamines slightly decreased the T m by 15 1.9–2.7 °C. Gly exhibited the best results, judging by the increased T m . The decreased T m value, as conferred by polyamines, implies that they bind to the unfolded mole- cules. DH and DC p values were approximately the same (within 5%) in the presence or absence of additives. These results suggest that the thermodynamic equilibrium of lysozyme is not influenced by 100 m M additive and that the molecular mechanism of spermidine and spermine as aggregation suppressors cannot be explained by the slight change in the thermodynamic parameters. Table 1. Residual activity of lysozyme after heat treatment. Additive Residual activity (%) a Residual activity (%) b Putrescine 15.1 ± 2.4 67.6 ± 4.8 Spermidine 52.4 ± 5.0 82.2 ± 6.1 Spermine 56.6 ± 4.3 90.5 ± 4.7 Lysine 2.3 ± 0.4 32.1 ± 6.6 Arginine 4.3 ± 1.3 30.9 ± 7.1 Glycine 1.5 ± 1.2 15.4 ± 2.8 Guanidine 0.8 ± 0.8 23.5 ± 2.9 Urea 0.8 ± 0.3 11.8 ± 3.1 NaCl 2.3 ± 0.4 13.2 ± 1.9 KCl 1.0 ± 0.7 11.8 ± 2.8 Glucose 0.9 ± 0.6 11.8 ± 1.3 Maltose 1.5 ± 0.7 13.1 ± 0.9 No additive 0.8 ± 0.8 21.3 ± 4.1 Before heating 100.0 ± 4.8 100.0 ± 6.1 a Residual activities of 0.2 mgÆmL )1 lysozyme containing 100 m M additive (pH 6.5) after heat treatment at 98 °C for 30 min. b Residual activities of 1.0 mgÆmL )1 lysozyme containing 100 m M additives (pH 6.5) after heat treatment at 98 °C for 10 min. Fig. 5. Differential scanning calorimetry (DSC) measurement. The samples contained 4.0 mgÆmL )1 lysozyme and 100 m M additive in 50 m M sodium-acetate buffer (pH 4.4). No additive, s;arginine(Arg), h;putrescine,n; spermidine, ,. Ó FEBS 2003 Prevention of thermal aggregation by polyamines (Eur. J. Biochem. 270) 4551 Discussion The present study indicates two points regarding the heat- induced aggregation and inactivation of lysozyme, namely (a) in the absence of additives, loss of activity is dependent on the protein concentration (Fig. 4A), indicating that the rate-limiting step of the heat inactivation of lysozyme is involved in an intermolecular interaction and (b) the resolved and refolded samples did not increase the activity (Fig. 4A). These two facts imply that the heat-induced inactivation of lysozyme is caused by covalent interactions among molecules, probably disulfide reshuffling. Interest- ingly, in the presence of spermidine and spermine, the inactivation rates were not dependent on the protein concentration (Fig. 4B,C). This implies that spermidine and spermine prevent intermolecular interactions. More- over, after heat treatment at 98 °C for 30 min, no aggre- gates were observed in the presence of 100 m M spermidine or spermine (Fig. 2A), while 50% of the molecules were inactivated (Fig. 4B,C). These facts propose the following mechanism, whereby the heat-induced aggregation and inactivation of lysozyme is considered to follow two steps of an irreversible reaction at high temperatures: U ! A Eqn ð1Þ A þ A n ! A nþ1 Eqn ð2Þ where, U represents the unfolded molecule that can refold after heat treatment, A represents the irreversibly denatured molecule and A n represents the insoluble aggregates. Under Eqn (1), spermidine and spermine prevent the intermolecular interactions shown in Eqn (2). As shown by Klibanov and co-workers [23,28], all proteins inactivate by heat; however, spermidine and spermine prevent heat inactivation of lysozyme as a result of inhibiting intermolecular interactions – the rate-limiting step. Some research has reported that the heat inactivation of proteins is caused by both noncovalent and covalent modifications, including disulfide exchanges [23,24,28], b-elimination of disulfide bonds [24,28], and deaminations of Gln and/or Asn [25,28]. At neutral pH values, the rate- limiting step of covalent modification is disulfide exchange [23,24,28]. Volkin & Klibanov showed that the half-lives of destruction of the disulfide bonds in 14 proteins at 100 °C were 0.6–1.4 h at pH 8 and 9–16 h at pH 6; lysozyme was no exception for the inherent thermal instability of disulfide bonds [ 16 23]. In view of these facts, we conclude that spermidine and spermine prevent intermolecular inter- actions, including disulfide exchanges and aggregation 17 . During early studies on protein aggregation, it was found that denaturing reagents of tertiary structures, such as guanidine and urea, increased the solubility of protein and improved the yield of refolding [10,11]. Other denaturing substances, such as lauryl maltoside micells [29] and surfactants [30], were found to promote the correct folding of proteins. When using these additives it is important to use the appropriate nondenaturing con- centration, but this may be difficult because the native state is easily destabilized in the presence of these additives. 18 On the other hand, Arg has a favorable property – it is not a denaturant, yet it enhances the solubility of the aggregate-prone form of unfolded protein [10,12,31]. For this reason, Arg has been commonly used as an aggregation suppressor. However, we report, in this study, the new finding that spermine and spermidine are more effective for preventing heat-induced aggregation than other, well-known additives (Table 1). Many researchers have reported the biological role of polyamines in enhancing growth or cell proliferation [32,33]. Polyamines are relatively simple structures that are com- posed of multivalent amines. The pK a values of the secondary amines in putrescine, spermidine, and spermine were 8.0–8.5, whereas those of the primary amines were 10.0–11.1 [34]. In biophysical aspects, polyamines can bind with nucleic acids and phospholipids, and stabilize and regulate their tertiary structures [17–19]. In this article, we report that polyamines prevent aggregation of lysozyme, a positively charged protein 19 (pI ¼ 11). Under acidic condi- tions, lysozyme can recover its active form, even after heat treatment for several hours [23,25], suggesting that some degree of additional charge neutralization may be at work. Although the present report did not investigate the precise mechanism of formation and inhibition of aggregates, our data imply that the formation of ion pairs with local negative charges would effectively increase the net charge of the protein, leading to increased electrostatic repulsion and Table 2. Thermodynamic parameters of lysozyme with additives. DCp, heat capacity change; DH, enthalpy change; T m , midpoint temperature of thermal unfolding. Thermodynamic parameters were determined by differential scanning calorimetry measurement of 4.0 mgÆmL )1 lysozyme with 100 m M additives in buffer. Additive T m at pH 6.5 (°C) a T m at pH 4.4 (°C) b DH at pH 4.4 (kJÆmol )1 ) b DC p at pH 4.4 (kJÆmolÆK )1 ) b No additive 77.3 81.0 452 20.7 Putrescine 78.4 79.1 436 21.7 Spermidine 78.4 78.7 437 22.2 Spermine 78.6 78.3 448 22.8 Arginine 77.8 79.7 439 21.6 Glycine 77.7 81.3 442 20.6 Guanidine 77.1 79.1 437 22.0 NaCl 77.7 79.9 441 21.6 a 50-m M sodium-phosphate buffer (pH 6.5); b 50 m M sodium-acetate buffer (pH 4.4). 4552 M. Kudou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 a reduction of intermolecular interaction. It is worth mentioning that hyperthermophiles, which can grow at temperatures of > 90 °C, synthesize several kinds of multivalent polyamines as their culture temperature increa- ses [14–16]. Our results imply that polyamines play a significant role in preventing heat-induced aggregation and inactivation of proteins in vivo. In conclusion, our results indicate that polyamines are a new class of additives which can prevent the aggregation and inactivation of heat-labile proteins. We propose that the following two questions should be addressed during future investigations of the efficacy of spermidine and spermine on protein aggregation. 20 First, can polyamines prevent the aggregation and inactivation of other proteins? Although it is still unclear, preliminary data has been obtained that heat inactivation of trypsin is effectively inhibited by polyamines. Second, can polyamines prevent the formation of 21 other types of aggregates, such as fibril formation or refolding- induced aggregation? Recently, Hoyer et al. reported that polyamines induce fibril aggregation of a-synuclein [35]. Our preliminary data using a model peptide reached the same conclusion. 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