Journal of Protein Chemistry, Vol. tl, No. 2, 1992

Stability of Fungal a-Amylase in Sodium Dodecylsulfate Tsutomu Arakawa, Lz Lynne Hung, ~ and Linda O. Narhi ~

Received September 3, 1991

Unfolding of a fungal a-amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the a-amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for i0 rain, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 rain at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the a-amylase is weak; However~ the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the a-amylase is a slow process. KEY WORDS: Unfolding of a-amylase; SDS resistance of a-amylase; melting of SDS-amylase complex; SDS-PAGE.

1. I N T R O D U C T I O N 3

mechanism of their proteolytic activity in the presence of SDS, and concluded that these proteases unfold very slowly in aqueous SDS solutions and that the unfolding is facilitated by increasing SDS concentrations and temperatures (Narhi et al., 1988; Narhi and Arakawa, 1989; Kolvenbach et al., 1990). Since the increase in SDS concentration does not change the concentration of free SDS molecules, the above results indicated that the unfolding of the proteins occurs through interactions with SDS micelles, rather than through interaction with the free molecules. When the proteins are not unfolded in SDS, they show abnormal migrations in SDS-PAGE, while the unfolded forms migrate to their corresponding molecular weights. We are interested in other proteins which might exhibit behavior in SDS solutions similar to that described above. Isemura and Takagi (1959) have observed that Taka-amylase A shows an abnormal migration in the presence of SDS as analyzed by moving boundary electrophoresis, and ascribed the

Many proteins are unstable in the presence of sodium dodecylsulfate (SDS) and denature even at room temperature upon exposure to SDS. However, there are some proteins resistant to denaturation by SDS. Among them are the subtilisins which, because of this stability, are included in detergent formulations (Weber et al., 1972, Narhi and Arakawa, 1989; Narhi et al., 1988, 1991). Several other proteinases, such as proteinase R, T, and K belong to this class (Hilz et al., 1975; Kolvenbach et al., 1990; Samal et al., 1989a, b, 1990). These proteases are active in SDS, but lose activity with time, in particular at higher SDS concentrations. We have undertaken a study to elucidate the

Amgen Inc., Amgen Center, Thousand Oaks, California 91320. 2 To whom all correspondence should be addressed. Abbreviations used: SDS, sodium dodeeylsulfate; PAGE, polyacrylamide gel electrophoresis; fl-ME, fl-mereaptoethanol; GPC, gel permeation chromatography; CD, circular dichroism.

111 0277-8033/92/0400-0111506.50/0~, 1992PlenumPublishingCorporation

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observed behavior to the resistance to unfolding. Therefore, we have applied the SDS-PAGE analysis to study the stability of various a-amylases against SDS-induced denaturation and found that the fungal a-amylase shows peculiar behavior. This paper reports the results of unfolding of the fungal a-amylase by SDS. 2. MATERIALS AND M E T H O D S A fungal a-amylase was obtained from Sigma (catalog no. A-0273, Type X-A). The protein was mixed with 3 volumes of sample buffer (for final concentration of 1% SDS, 20% glycerol, 0.16% bromophenol blue) with or without fl-ME (2.5%). For GPC

and CD experiments, bromophenol blue was not included. SDS-PAGE was carried out using the Laemmli system (Laemmli, 1970) and 10% polyacrylamide gels. Protein was visualized by staining with Coomassie blue. The destained gels were scanned on an LKB Ultroscan XL laser densitometer to quantitate the amount of protein present in the various species. Circular dichroic spectra were determined using a Jasco J-500C spectropolarimeter controlled by a Samtron computer. Cuvettes with pathlengths of 1 cm were used for the near UV and 0.02 cm for the far UV. The spectra of fungal amylase mixed with sample buffer as described above were determined following incubation at room temperature, and after a 10 rain

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Fig. 1. SDS-PAGE of fungal a-amylaseincubatedwith 1% SDS at room temperature or 90°C. Lane l, no fl-ME and 90°C; lane 2, no fl-ME and room temperature; lane 3, fl-ME and 90°C; lane 4, fl-ME and room temperature.

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incubation at 90°C followed by 1 hr at room temperature. The spectra were recorded at ambient temperature. GPC analysis was performed on a Superose 12 column using a Pharmacia fast protein liquid chromatography system. The amylase was prepared as described above for the CD analysis and injected onto the column equilibrated in 10 mM sodium citrate, 0.75% SDS, 100 mM NaCI, pH 7, at a flow rate of 0.5ml/min. Fractions were collected and subsequently analyzed by SDS-PAGE.

3. RESULTS AND DISCUSSION

The stability of the fungal a-amylase in SDS was examined by mixing the proteinwith reducing or nonreducing SDS-PAGE sample buffer and incubating the mixture at room temperature or 90°C for 10 min. The mixtures were analyzed by SDS-PAGE on 10% polyacrylamide gels, as shown in Fig. 1. In the absence of fl-ME and heat, the a-amylase had a much greater mobility (lane 2) than expected for a protein with a Mr = 50,000 (Takagi et aL, 1971). Upon heating, the mobility of the protein was decreased (lane 1), but

was still greater than that of a protein of Mr = 50,000. When the protein was incubated in the presence of tiME at room temperature (lane 4), it migrated to a position identical to that of the sample incubated at room temperature without fl-ME, suggesting that under these experimental conditions the disulfide bonds are not reduced. Upon heating in the presence of fl-ME, the mobility of the protein decreased further (lane 3), and the sample migrated to the position expected for the Mr of 50,000, indicating that the disulfide bonds were cleaved. The observed abnormal mobility for the unheated samples suggests that the proteins were not unfolded by SDS. The slower migration of the heated samples suggests that the proteins were unfolded by SDS and heat. In the latter experiment, the faster migration of the sample without fl-ME relative to that with fl-ME is expected, since the disulfide bonds should reduce the hydrodynamic size of the protein-SDS complex relative to the reduced protein-SDS complex. To confirm the above conclusion, the conformation of the protein in SDS was analyzed by CD and GPC. Figure 2 shows the CD spectra of the protein incubated with 1% SDS at room temperature (dotted line). Both near and far UV spectra of the above sample were identical to those obtained in the absence of SDS (solid line), indicating that the protein retains the native secondary and tertiary structures. When heated at 90°C for 10 min, the sample lost the CD signal in the near UV region (broken line), as shown in Fig. 2, indicating that the combination of heat and SDS caused the amylase to unfold. The secondary structure of the sample was also altered in a manner consistent with an unfolded protein (broken line in lower panel); the a-helical content increased as expected for a protein-SDS complex. GPC experiments were performed in the presence of 0.75% SDS, since the column is not stable in 1% SDS; however, 0.75% SDS is well above the critical micelle concentration. In addition, the samples were treated with 1% SDS. Figure 3 shows the elution peaks of the unheated (solid line) and heated (dotted line) samples, displaying a much smaller elution volume for the heated sample. SDS-PAGE analysis of the fractions in the peaks revealed a band with the mobility of the folded protein in the unheated sample, while the peak fractions in the heated sample had the mobility of the unfolded protein. The results of the CD and GPC experiments demonstrate that the fungal a-amylase retains its native conformation in SDS and becomes unfolded only when heated.

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Fig. 5. Time course of unfolding of fungal a-amlyase in 1% SDS. (A) SDS-PAGE of the protein incubated at 50°C. Lanes 1 and 2 contain amylase heated at 90°C for 5 min; lane 3 contains amylase incubated at room temperature for 180 rain. The rest of the amylase shown was incubated at 50°C for 20 rain (lane 4), 40 rain (lane 5), 60 min (lane 6), 80 rain (lane 7), 100 min (lane 8), 120 min (lane 9), 140 rain (lane 10), 160 rain (lane 1l), and 180 rain (lane 12). (B) Ratios of staining intensity of the unfolded band to the folded state. The protein was incubated at 37°C (x), 50°C (O), and 60°C (FI).

Stability of Fungal a-Amylase The above results indicate that the unfolding of the protein by SDS is temperature dependent. Therefore, the a-amylase was incubated at different temperatures with 1% SDS for 10 rain and analyzed by SDS-PAGE, and the results are shown in Fig. 4A. The higher mobility band disappears and the (smearing) lower mobility band appears as the temperature is increased. The staining intensity of these two bands was densitometrically determined and the ratio of the staining intensity of the two bands was plotted against temperature, as shown in Fig. 4B. From this, the midtransition temperature was determined to be about 55°C. Similar experiments were carried out in the presence of 0 and 2% SDS, and the results are also plotted in Fig. 4B. Both plots show a reasonable transition pattern, with mid-transition temperature of 49 and 70°C, respectively, for 2% and 0% SDS experiments. The observed small differences in melting temperature between the different SDS concentrations suggest that the effect of SDS on the stability of the protein is weak. It should be pointed out, however, that in the presence of SDS the thermal transition may not be an equilibrium transition; the previous study with subtilisin showed that the protein fully unfolds at a rate depending on the temperature. The unfolding of the protein in 1% SDS was therefore examined at several temperatures as a function of time. At 37°C, no unfolding was observed following 6 hr in SDS (see Fig. 5). At 50°C, a gradual conversion of the higher mobility band to the lower mobility one was observed with time, as shown in Fig. 5A. Figure 5B shows the time course of the unfolding,

117 as determined from the staining intensity of the two bands. Unfolding is already seen after 20 rain and increases with time, reaching 70% at 2 hr. At 60°C, the unfolding occurs much faster, reaching nearly 100% at 40 rain. These results indicate that unfolding of the aamylase is a stow process, and is facilitated by higher temperature. All these experiments (except the experiments described in Fig. l) were carried out in the absence of fl-ME. The inclusion of fl-ME facilitates unfolding, as expected from the stabilizing action of disulfide bonds on the conformational unfolding of proteins. REFERENCES Hflz, H., Wiegers, U., and Adamietz, P. (1975). Eur. J. Biochem. 56, 103-108. lsemura, T., and Takagi, T. (1959). J. Biochem. 46, 1637 1644. Kolvenbach, C. G., Narhi, L. O, Langley, K., Samal, B., and Arakawa, T. (1990). ha. J. Peptide Prote#7 Res. 36, 387-391. Laemmli, U.K. (1970). Nature 227, 680-685. Narhi, L. O., and Arakawa, T. (1989). Biochim. Biophys. Acta 990, 144 149. Narhi, L. O., Stabinski, Y., Levitt, M., Miller, L. Sachdev, R., Finley, S., Park, S.0 Kolvenbach, C., Arakawa, T., and Zukowski, M. (1991). Biotech. Appl. Biochem. 13, 12-24. Narhi, L. O., Zukowski, M., and Arakawa, T. (1988). Arch. Biochem. Biophys. 261, 161-169. Samal, B. B., Karan, B., Boone, T. C., Chen, K. K., Rohde, M. F., and Stabinski, Y. (1989a). Gene 85, 329-333. Samal, B. B., Karan, B., Boone, T. C., Osslund, T. O., Chert, K. K., and Stabinski, Y. (1990). Molec. Microbiol. 4, 1789-1792. Samal, B. B., Karan, B., and Stabinski, Y. (1989b). Biotech. Bioengineer 35, 650-652. Takagi, T., Toda, H., and Isemura, T. (1971). In The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. V., Academic Press, New York, pp. 235-271. Weber, K., Pringle, J. R., and Osborn, M. (1972). Methods Enzymol. 26, 3-27.