Tài liệu Báo cáo khoa học: Arginine ethylester prevents thermal inactivation and aggregation of lysozyme pptx

Arginine ethylester prevents thermal inactivation and aggregation of lysozyme Kentaro Shiraki 1 , Motonori Kudou 1 , Shingo Nishikori 1,2 , Harue Kitagawa 1,2 , Tadayuki Imanaka 3 and Masahiro Takagi 1,2 1 School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), Tatsunokuchi, Japan; 2 Innovation plaza Ishikawa, Japan Science and Technology Agency (JST), Tatsunokuchi, Japan; 3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan Arginine is a versatile additiv e to prevent p rotein aggrega- tion. This paper shows that arginine ethylester (ArgEE) prevents heat-induced inactivation and agg regation o f h en egg lysozyme more effectively than arginine or guanidine. The addition of ArgEE decreased the melting temperature o f lysozyme. This data could be interpreted in terms of A rgEE binding to unfolded lysozyme, possibly through the ethyl- ated carboxyl group, which leads to effective prevention of intermolecular interaction a mong aggregation-prone mole- cules. The data suggest that ArgEE could be used as an additive to prevent inactivation and aggregation of heat- labile proteins. Keywords: arginine; arginine ethylester; lysozyme; protein aggregation; thermal inactivation. Protein a ggregation is a serious problem for both biotech- nology and cell biology. Diseases such a s prion misfolding, Alzheimer’s, and other amyloidoses are phenomena f or which protein aggregation in our living cells is of consid- erable relevance [1–4]. In the field of biotechnology, aggregation, resulting in the formation of inclusion bodies, is a major problem in bacterial recombinant systems [5–7]. Under unfolding conditions, irreversible a ggregation competes with correct folding. The classical model by Lumry–Eyring has b een used to describe protein aggrega- tion [8–10]: N $ A ! Agg ð1Þ where N, A, and Agg represent a native state, a non-native state, and aggregates. Equation (1) involves a first-order reversible folding/unfolding reaction and subsequent inter- molecular association w ith a higher-order irreversible pro- cess. The kinetics and equiliblium of Eqn (1) are dependent on solution conditions, such as temperature, pH, and the presence of additives. The additives may influence both the solubility and the stability o f proteins in the N a nd A states. They also may change the folding rate to prevent or accelerate the nonspecific aggregation from A to Agg. Guanidine and urea are well established as aggregation suppressors that weaken the hydrophobic intermolecular interaction of p roteins [11,12]. In particular, these denatu- rants increase the solubility of aggregation-prone unfolded molecules, but decrease the s tability of the native state. Among nondenaturing reagents, a rginine is t he most widely used additive for increasing refolding yields by decreasing aggregation, for example when it is used in experiments wit h a single chain antibody [11,13]. Arginine does not facilitate refolding, but suppresses aggregation, with only a minor effect on protein stability [14], while it enhances the solubility of aggregates-prone molecules, leading to an increase in refolding yields [15–17]. Although other addi- tives, such as proline, glycerol, glycine, and ethylene glycol, have been used [12], these are not enough t o solve the problems of protein aggregation and misfolding. Recently we reported that polyamines, typically spermine and spermidine, prevent heat-induced inactivation and aggre- gation of lysozyme [18,19]. As p art of a series of studies to develop additives, this paper shows a new candidate, arginine ethylester (ArgEE), as a superior additive to prevent heat-induced inact ivation and aggregation of lyso- zyme as a model protein. Materials and methods Materials Bovine pancreas RNaseA, hen egg white lysozyme, horse myoglobin, Arginine/HCl (Arg), and A rgEE were from Sigma Chemical Co. Guan idine h ydrochloride (GdnHCl), NaCl, Na 2 HPO 4 ,andNaH 2 PO 4 were from Wako Pure Chemical Industries Ltd. All chemicals used were of high quality analytical grade. Time course of thermal inactivation and aggregation Heat treatment o f lysozyme w as performed a s f ollows: 500 lL of the sample solutions containing 1.0 mgÆmL )1 or 0.2 m gÆmL )1 lysozyme and 100 m M sodium phosphate buffer pH 7.1 in the presence or absence of 100 m M Correspondence to K. Shiraki, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan. Tel.: +81 761 51 1657, E-mail: kshiraki@jaist.ac.jp Abbreviations: ArgEE, arginine ethylester; Gdn, guanidine; T m , midpoint temperature of thermal unfolding. Enzymes: lysozyme (EC 3.2.1.17); ribonuclease A (EC 3.1.27.5). (Received 4 April 2004, revised 7 June 2004, accepted 17 June 2004) Eur. J. Biochem. 271, 3242–3247 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04257.x additives were prepared in 1.5 mL microtubes. The samples were heat treated at 98 °C for various periods. After the heat treatment, th e samples were immediately cooled on ice for 4 h. The samples were centrifuged at 15 000 g for 20 min at 4 °C, and then the concentration o f soluble protein and residual activity were determined. The protein concentration o f the supernatants was determined by measuring absorbance at 280 nm with the appropriate blank, using e xtinction co efficients of 2.63 cm )1 per mgÆml )1 . Measurements of protein concentration and residual activity The concentration of soluble protein was monitored with a Jasco spectrophotometer model V-550 (Japan Spectro- scopic Company), using an extinction coefficient of 2.63 cm )1 per mgÆml )1 [20]. The residual activity of the soluble fraction was determined as follows [9,18]: 1.5 mL of 0.5 mgÆmL )1 Micrococcus lysod eikticus solution contain ing 50 m M sodium phosphate buffer pH 6.5 was mixed with 20 lL o f the protein solution. The decrease in light scattering intensity of the solution was monitored by absorbance at 600 nm. The re sidual activity was e stimated by fitting the data to a linear extrapolation. pH dependence of thermal inactivation and aggregation Heat treatment of lysozyme was performed according to t he following procedure: 500 lL of the sample solutions containing 1.0 mgÆmL )1 lysozyme and at variou s pH values (adjusted by t he addition of 100 m M phosphate borate buffer) in the presence or absence of Arg or ArgEE were prepared in 1.5 mL microtubes. The samples were heated at 98 °C for 10 min. After the heat treatment, the samples were cooled on ice for 4 h. The samples w ere centrifuged at 15 000 g for 20 min at 4 °C, and then the concentration of soluble protein and residual activity were determined. After these measurements, the precise pH was d etermined using the residual sample. Thermal unfolding by DSC Thermal unfolding curves of lysozyme were measured by DSC, using a nano-DSCII differential scanning calorimeter 6100 (Calorimetry Sciences Corporation) with a cell v olume of 0.299 m L at a scanning rate of 2 °CÆmin )1 . Degassing during the calorimetric experiment was prevented by maintaining an additional constant pressure of 2.5 bar over the liquid in the cell. Solution contained 4.0 mg ÆmL )1 lysozyme, various concentrations of Arg or ArgEE, and 100 m M sodium phosphate buffer pH 6.5. The apparent melting temperature ( T m ) was determined at the peak of the DSC curve. Thermal unfolding by near-UV CD Thermal unfolding curves of myoglobin and RNaseA were measured by near-UV CD, with a Jasco spectropolarimeter model J-720 W equipped with t hermal incubation system. The samples containing 1.0 m gÆmL )1 protein, 500 m M additive, and 100 m M sodium phosphate buffer pH 6.5 were prepared. The thermal unfolding was measured by CD at 280 nm intensity with increasing temperature of 1 °CÆmin )1 . The data obtained w ere fitted to a conventional two-state equation and determined the apparent T m . Results Thermal inactivation and aggregation of lysozyme in the presence of additives Figure 1 s hows the thermal inactivation and aggregation of lysozyme at pH 7.1. In the absence of any additives, lysozyme was inactivated and a ggregated with a single- exponential manner after a lag period of  200 s (Fig. 1A). The presence of lag phase on the inactivation curve implies that the inactivation is affected by the formation of aggregates during heating. This is consistent with previous data showing that the thermal inactivation of lysozyme has a single rate-limiting step [21–25]. Actually, more than 10 types of protein have been analysed, s howin g that the higher-order processes of aggregates can be described by single-exponential equations [26,27]. In the presence of 100 m M Arg, the inactivation and aggregation rates were slightly decelerated (Fig. 1B). In the presence of ArgEE, the heat-induced inactivation rate of lysozyme was one-sixth that in the absence of additives (Fig. 1C). The rates of inactivation and aggr egation under several different conditions are shown in Table 1. The rates o f inactivation and aggregation d epend on the protein Fig. 1. Thermal inactivation and aggregation of lysozyme in the presence of additives. T he samples containin g 1.0 mgÆmL )1 lysozyme with no additive (A), 100 m M Arg (B), and 100 m M ArgEE (C) at pH 7.1 were h eated a t 9 8 °C for the times shown. After t he heat treatment, the residual activity (s) and the amount o f aggregate c alculated by the concentration of solub le protein ( d) were determined and p lotted. The c ontinu ous and broken lines show the theoretical curves fitted to the closed and open circles with single exponen tial equations. Ó FEBS 2004 Aggregation of lysozyme with additive (Eur. J. Biochem. 271) 3243 concentration, indicating that intermolecular interaction is the rate-limiting step in both inactivation and aggregation of lysozyme by heat treatment. As the d ata points were well fitted to the single-exponential eq uation, the heat- induced aggregation and inactivation of lysozyme appar- ently follow first-order kinetics. However, the fact that the rates of aggregation and inactivation depend on protein concentration allows us to consider the processes as pseudo-first order, as reported previously [18,26,27]. These data imply that the rate-limiting step of aggregation and inactivation is the stage of irreversible unfolding, which is affected by the additives. After the obligatory process of unfolding, t wo (or several) p rotein molecule s transform the a ggregation-prone un folded molecules to t he aggre- gates. ArgEE lowered the dependence of the rate of aggregation on protein concentration, implying that ArgEE prevents intermolecular interactions. The rates of inactivation and aggregation of lysozyme in the presence of ArgEE were similar to those in spermine, which is a favourable additive to prevent t hermal inactivation and aggregation of l ysozyme [18]. These data s how that ArgE E is a new candidate additive for the prevention of thermal inactivation of lysozyme. pH dependence of the inactivation and aggregation in the presence of additives Figure 2 shows the pH-dependent inactivation and aggre- gation of lysozyme. After heat treatment at 98 °Cfor 10 min, 1.0 m gÆmL )1 lysozyme without additives was completely inactivated above pH 7.2 (Fig. 2A, s). Several reports of the heat-induced inactivation of lysozyme have shown that t he inactivation at alkaline pH is highly related to the intermolecular noncovalent interactions, followed by covalent modification, mainly caused by disulfide exchange [21,22,24]. This is because the pI of lysozyme is around pH 11. After the sa me heat trea tment, the sigmoidal a ctivity curve was slightly improved by the addition of 100 m M Arg (Fig. 2 A, h). On the other hand, the sigmoidal activity curve was clearly shifted to alkaline p H by the addition of 100 m M ArgEE ( Fig. 2A, n). For example, 80% or more of the enzymes retained the active form after heat treatment at 98 °C for 10 min in the presence of 100 m M ArgEE at pH 6.5; under the same conditions, only 30% of the enzymes retained the active form even in the presence of the same concentration of Arg. ArgEE p revents heat-induced aggregation (Fig. 2B) as well as inactivation (Fig. 2A). After heat t reatment at 9 8 °C for 10 min, the amount of aggregates increased with increasing pH (Fig. 2B, s). In the p resence of 100 m M Arg, the profile was slightly improved. However, in the presence of 100 m M ArgEE, the p rofile was clearly shifted to alkaline pH (Fig. 2B, n). Interestingly, in the presence of 1.0 M NaCl, the inacti- vation and agg regation c urves i n the presence of Arg are the same as those in the absence of additives (Fig. 2C,D). Th is indicates that t he prevention of inactivation and aggregation by Arg can be explained solely by electrostatic interactions. On the basis that heat-induced aggregation is due to the intermolecular interactio n between exposed hydrophobic regions, Arg may play a role in the prevention of intermolecular interactions due to electrostatic interaction s. On the other hand, the inactivation and aggregation curves obtained with 100 m M ArgEE are clearly different in the presence of 1.0 M NaCl; ArgEE prevents both t hermal inactivation and aggregation at high p H (Fig. 2B,D). The Table 1. Rates of thermal inactivation and aggregation in the presence of additives. Thermal inactivation and aggregation in the presence or absence of 100 m M additive were measured as shown i n Fig. 1 . The inactivation and aggregation profiles were fitted to single exponential equations and th e a pparent r ate c onstants w ere c alculated. n d, N o data due to the slow rate of aggregation under the conditions used. Protein concentration Additive Inactivation (· 10 )3 Æs )1 ) Aggregation (· 10 )3 Æs )1 ) 1.0 mgÆmL )1 (pH 7.1) None 7.04 ± 0.56 4.42 ± 0.43 NaCl 5.85 ± 0.24 4.54 ± 0.23 GdnHCl 4.81 ± 0.44 2.61 ± 0.38 Spermine 1.22 ± 0.13 0.57 ± 0.14 Arg 4.24 ± 0.39 2.17 ± 0.25 ArgEE 1.15 ± 0.15 0.42 ± 0.08 0.2 mgÆmL )1 (pH 7.1) None 4.03 ± 0.24 1.11 ± 0.06 Arg 1.62 ± 0.25 1.09 ± 0.06 ArgEE 0.84 ± 0.11 0.35 ± 0.08 0.2 mgÆmL )1 (pH 6.5) None 1.01 ± 0.09 0.41 ± 0.21 Arg 0.76 ± 0.16 0.28 ± 0.17 ArgEE 0.11 ± 0.03 nd Fig. 2. pH-dependent thermal inactivation and aggregation of lysozyme in the presence of additives. Samples containing 1.0 mgÆmL )1 lysozyme and 0 M (A,B) or 1.0 M (C,D) N aCl w ith 100 m M additives at vario us pHswereprepared.Theadditivesarenone(s), Arg (h), or ArgEE (n). These samples were heated at 98 °C for 10 min and residual activity (A,C) and amount of aggregates (B,D) were determined. Continuous, dotted, and broken lines show the fitted curves to no additives, Arg, and ArgEE with sigmoidal equations. 3244 K. Shiraki et al. (Eur. J. Biochem. 271) Ó FEBS 2004 data obtained in the presence of NaCl suggest that the molecular mechanism of ArgEE in preventing thermal inactivation and aggregation is different from that of Arg. Thermal unfolding profile of proteins in the presence of ArgEE In order to investigate whether or not ArgEE destabilizes protein structure, we analysed the thermal unfolding profile of lysozyme in the presence of additives. Figure 3A shows the thermal unfolding curve of lysozyme in the presence of ArgEE as m onitored by DSC. In the absence of a dditives, the T m value of lysozyme w as 78.1 °CatpH6.5.TheT m increased a s the concentration of ArgEE increased from 0 to 60 m M . The maximum T m of lysozyme is 79.8 °Cat60m M ArgEE. The increase in T m may correspond to the increase in solubility of t he aggregation-prone molecules caused by the addition of ArgEE. With further increases in the concentration of ArgEE, the T m decreased from 100 to 600 m M . The decreasing T m corresponds to the unfolding effect of ArgEE on lysozyme. Figure 3B summarizes the T m of lysozyme in the presence of Arg and A rgEE. Unlike with ArgEE, the T m did not change with increasing concentra- tions of Arg. Figure 4 shows thermal unfolding curves of RNaseA (Fig. 4 A) and myoglobin (Fig. 4B) in the presence or absence of additives as monitored by near-UV CD. The T m value of RNaseA in the absence of additives was 64.7 ± 0.1 °C. In the presence of 500 m M GdnHCl and Arg, T m values of RNaseA were 59.6 ± 0.1 °Cand 60.8 ± 0.2 °C, respectively, which were 5.1 °Cand3.9°C lower than those obtained without the a dditives. The T m of RNaseA with 50 0 m M ArgEE (56.1 ± 0.2 °C) was 8 .6 °C lower than without additives. Similarly, the T m of myoglo- bin in the presence of 500 m M ArgEE (58.3 ± 0 .4 °C) w as clearly lower than in the presence of 500 m M Arg (74.2 ± 0.4 °C). These data, being consistent with the DSCanalysesoflysozyme,suggestthatArgEEhasa destabilizing effect on proteins. The thermal unfolding curves of myoglobin in the absence of a dditive and in the presence of GdnHCl could not measured by near-UV CD due to the aggregation that occurred under these conditions. An identical experiment was performed with lysozyme but the near-UV CD data generated were too noisy to allow evaluation of the T m . Charged states of Arg and ArgEE In order to understand the importance of amphiphilicity, titration curves of Arg and ArgEE were compared (Fig. 5 A). The pK a of amino groups on Arg and ArgEE were determined as pH 9.2 and 7.4, respectively. The decreased pK a of amino group of ArgEE compare to Arg is related to the ethylation of the main chain of the carboxyl group. Figure 5B shows the amount of aggregation of lysozyme at pH 6.5 and 10.0 in the presence of Arg and ArgEE after heat treatment a t 98 °C for 30 min. At pH 6.5 Arg and ArgEE possess positive charges on their amino group, while at pH 10.0 they lose the positive charges. W ith increasing concentration of ArgEE, the amount of aggre- gates steeply decreased from 0 to 30 m M . The addition of 30 m M ArgEE completely prevents heat-induced aggrega- tion of lysozyme at pH 6.5. The preventive effect of ArgEE was clearly higher than that of Arg (Fig. 5B). However, no Fig. 3. Thermal unfolding cu rves of ly so zyme in th e p rese nce o f additiv es monitoredbyDSC.The samples containing 4.0 mgÆmL )1 lysozyme with various con centrations of Arg or ArgEE at pH 6.5 were measured by DSC. (A) Representative curves in the presence of ArgEE. The concentrations of ArgEE were shown in the figure. (B) T m in the presence of Arg (d)orArgEE( s). Fig. 4. Thermal unfolding curves of proteins in the presence of add itives monitoredbynear-UVCD.The samples cont aining 4.0 mgÆmL )1 RNaseA (A) or myoglobin (B) in the presence or absence of 500 m M additive at pH 6.5 w ere measured by CD at 280 nm. The ad ditives are no additive ( s), Gd nHCl (h), Arg (n), and ArgEE (·). The data were fitted to conventional two-state equations. Fig. 5. Differences in the c hemi cal properties of Arg and ArgEE. (A) Determ ina tio n of p K a values of amino groups on Arg and ArgEE. A small q uantity of 1.0 M NaOH wa s added to 10 mL of 1.0 M Arg/HCl (s)orArgEE-2HCl(h) solution. (B) Heat-induced aggregation o f lysozyme in the presence of A rg (circles) or A rgEE (squares) at pH 6.5 (open symbols) or pH 10.0 (closed symbols). T he samples containing 0.2 mgÆmL )1 lysozyme with additives a t pH 6.5 or 10.0 were he ated at 98 °C for 30 min. The samples were centrifuged at 15 000 g for 30 min, and then the amount of aggregates was determined. Ó FEBS 2004 Aggregation of lysozyme with additive (Eur. J. Biochem. 271) 3245 prominent effect of ArgEE w as observed when m onitoring at pH 10.0 (Fig. 5B). These data suggest that ArgEE prevents heat-induced aggregation only in the charged state of the amino group. Discussion From early studies on protein aggregation, it is known t hat denaturing reagents, such as GdnHCl and u rea, increase the solubility o f a ggregate-prone molecule s, leading to i mprove- ment in refolding yield [12]. On the other hand, Arg is a nondenaturing reagent that prevents protein aggregation [14–16]. Although Arg is one of the m ost w idely u sed additives f or the prevention of p rotein aggregation a nd improvement of refoldin g yield, only a few papers have reported the molecular mechanism of Arg as an additive. The following properties are shown: (a) Arg is the best additive for the prevention of heat-induced aggregation of lysozyme out of 15 amino a cids [15] and ( b) Arg does not stabilize proteins against heat treatment [16]. In addition, this paper shows t hat (c) Arg p revents heat-induced aggregation by an e lectrostatic interaction between protein molecules (Fig. 2). This paper focused on the ArgEE as a new additive to prevent heat inactivation and aggregation. We selected ArgEE a s additive b ecause it i s an Arg derivative that possesses g uanidium group on its side c hain. Although we have examined several Arg derivatives, only ArgEE shows a strong effect in preventin g protein inactivation. The molecular mechanism of ArgEE in preventing heat-induced aggregation is different from that of Arg. ArgEE may bind preferentially to unfolded molecules of l ysozyme by the introduced hydrophobic e nd on the carboxyl grou p, leading to an increase in the apparent net charge of the unfolded molecules. The increased net charge caused by binding of the additives would effectively increase the electrostatic repulsion between unfolded or partially unfolded molecules that are prone to form irreversible aggregates and reduce aggregation and misfolding. Hen egg white lysozyme is inactivated irreversibly b y heat treatment a t n eutral pH [21–24]. The rate- limiting step of inactivation at around pH 7 is the intermolecular interaction between exposed hydrophobic surfaces, fol- lowed by irreversible disulfide exchange [24,25]. The irreversible aggregation of protein caused by heat treat- ment usually follows pseudo first-order kinetics at the terminal phase, such as seen with beef catalase [28], beef citrate synthase [29], bovine alpha A-crystallin [30], a nd ovalbumin [31], although the process of aggregation is expected to be second- or higher-order kinetics [8,32–34]. This is because t he rate-limiting s tep of thermal aggre- gation is th e nucleation with g rowth of aggregates, leading to p seudo-first-order kinetics after a lag period of  200 s. The p resence of t he lag phase observed i n both inactivation and aggregation results from the structural change to aggregation-prone molecules. The aggregation-prone molecules m ust possess low solubility and a large hydrophobic region on the surface in comparison with the soluble unfolded molecules [22,35,36]. Our data described by the pseudo-first-order kinetics also support the same conclusion even in the presence of Arg and ArgEE. 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Results Thermal inactivation and aggregation of lysozyme in the presence of additives Figure 1 s hows the thermal inactivation. The rates o f inactivation and aggregation d epend on the protein Fig. 1. Thermal inactivation and aggregation of lysozyme in the presence of additives.