Secondary structural changes of N-bromosuccinimide-cleaved bovine serum albumin in solutions of sodium dodecyl sulfate, urea, and guanidine hydrochloride

Secondary Structural Changes of N-Bromosuccinimide-Cleaved Bovine Serum Albumin in Solutions of Sodium Dodecyl Sulfate, Urea, and Guanidine Hydrochloride KUNIO

T A K E D A 1 AND A K I R A W A D A

Department of Applied Chemistry, Okayama Universityof Science, 1-1 Ridai-cho, Okayama 700, Japan Received August 27, 1990; accepted October 25, 1990 Two tryptophyl peptide bonds of bovine serum albumin (BSA), Trp134-Gly135and Trp212-Ser213, were cleaved with N-bromosuccinimide (cleaved BSA). The relative extents of secondary structures in cleaved BSA were determined by curve-fitting of the circular dichroism spectrum. Cleavage at these two sites caused an appreciable disruption in the helicity of the protein. The cleaved BSA contained 55% c~helical structure as against 66% for the intact BSA. The cleavagealso made the helical structures unstable in solutions of urea and guanidine hydrochloride. The extent of c~-helical structure of cleaved BSA decreased to 50% in sodium dodecylsulfate (SDS) solutions. However, some moieties, the helical structures of which had been disrupted by N-bromosuccinimide cleavage, appeared to re-form the helical structure in SDS, although the cleavage effect remained through the urea and guanidine denaturations and also through the thermal denaturation. On the other hand, the three resultant polypeptides, Asp ~-Trp 134, Gly~35-Trp2~2, and Ser2~3-Alas82, were separated by reduction of the disulfide bridges joining them. These three separated polypeptide fragments showed only 20% helix content. However, the extent of the helical structure in the three polypeptide fragments increased to 35-40% in SDS. Secondary structural changes in the three fragments were also examined in urea and guanidine hydrochloride solutions. © 1991 Academic Press, Inc.

sodium dodecyl sulfate (SDS) (6) and the binding isotherm o f SDS to the same fragments (7). In the present study, two sites, Trp 134-Gly 135 and TrpZlZ-Ser213, on the single polypeptide o f BSA were cleaved with N-bromosuccinimide (NBS) ( 1 ), the three resultant fragments being linked by disulfide bridges (this BSA is designated hereafter as cleaved BSA). The intact BSA has five large loops, each consisting o f two helical rods, joined at one end by a C y s - C y s bridge and the other by the polypeptide b a c k b o n e (1). The present cleavage brings about disruption o f two o f the five large loops. Three isolated fragments, A s p l - T r p TM, Gly135-Trp 212, and Ser 2~3Ala 582, were also obtained through the reduction o f the disulfide bridges in the cleaved BSA. Circular dichroism ( C D ) o f the cleaved BSA and the three isolated fragments in urea and guanidine hydrochloride as well as in SDS was

INTRODUCTION Bovine serum albumin (BSA) is c o m p o s e d o f 582 a m i n o acid residues in a single polypeptide chain with l 7 rather regularly distributed disulfide bridges. According to Brown, this protein has three domains, each consisting o f a large double loop, a short connecting segment, a small double loop, a long connecting segment ( H i n g e ) , another large double loop, and a connecting segment to the next d o m a i n ( l ). M a n y investigators have studied the conformation o f BSA in surfactant solutions ( 2 4). However, m o s t o f these studies have been concerned with the intact BSA molecule. Few attempts have been m a d e to study structural changes in fragments o f the protein (5, 6). Recently, we have studied secondary structural changes o f domain-sized fragments o f BSA in To whom correspondence should be addressed. 45

0021-9797/91 $3.00 Journal of Colloid and Interface Science, Vol. 144, No. 1, June 1991

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

46

TAKEDA

measured to examine the effect of cleavage on the secondary structure of the protein.

AND

WADA

duced BSA (12), this separation pattern indicates that NBS cleaves only two sites on the single BSA polypeptide and then the present cleaved BSA is separated into three fragments. EXPERIMENTAL Plural amino acid residues at the N and C Crystalline BSA and SDS were the same as terminals of the three fragments were analyzed used before (6, 7). NBS, urea, and guanidine as described previously (6). In the analysis of hydrochloride were purchased from Nacalai amino acid residues at C terminals, only the Tesque, Inc. detection of Trp required a pH gradient difThe use of NBS as a specific reagent for ferent from that for the other amino acids. It tryptophan in proteins has led to two major was finally confirmed that the three fragments applications: one is a rapid spectrophotometric corresponded to Asp l-Trp 134, Gly 135-Trp212, determination of the tryptophan content of a and Ser213-Ala 582 within the sequence of the protein (8); the other provides a means of parent protein, BSA. SDS-polyacrylamide gel cleaving the tryptophyl peptide bond ( 8-11 ). electrophoresis also gave molecular weights for Prior to cleavage of the BSA polypeptide by the fragments similar to those calculated from NBS, a free SH group of the protein was these sequences. In addition, the results of the masked with iodoacetamide (12). Cleavage at amino acid analysis indicate that only two Trp 134-Gly135 and TrpZ12-Ser213 in BSA was tryptophyl peptide bonds are cleaved in the carried out at 25°C for 6 h in a 0.2 M acetic present NBS cleavage. The cleavage sites are acid solution (pH 4.0) containing 8 M urea consistent with those reported previously [ see and NBS. The concentration of NBS was 10 Fig. 6 in Ref. ( 1)]. times the molar equivalent of the protein. In Polypeptide concentrations were detercleaved BSA, the three resultant fragment mined spectrophotometrically. Extinction polypeptides are linked by disulfide bridges. coefficients of cleaved BSA and the three fragThe fragment polypeptides were separated by ments were estimated on the basis of their reduction of the disulfide bridges in the cleaved concentrations determined by weighing the BSA. Dithiothreitol was used to reduce the di- lyophilized samples of 50-100 nag (6, 12). The sulfide bridges ( 12, 13). Subsequently, the samples had been exhaustively dialyzed cleaved BSA and the isolated fragments were against pure water to remove the electrolytes dialyzed against a phosphate buffer of pH 7.0 composing the buffer and then they were lyand then concentrated using a freeze-drier. ophilized. The extinction coefficients at 280 Their purifications were carried out with an nm were 45,000 for cleaved BSA [44,000 ion-exchange column, Mono Q HR-5/5 for the intact BSA ( 14)], 27,400 for Ser 213(Pharmacia), connected to a fast protein liq- Ala 582, 10,300 for Asp l-Trp 134,and 5,800 for uid chromatography (FPLC) system (Phar- Gly 1 3 5 - T r p 212 . macia). The cleaved BSA was further purified The isotherm for binding of SDS to the by passage through a Sephacryl S-200 (Phar- cleaved BSA was obtained at 25°C by gel macia) column. chromatography using a high-performance When reduction of the disulfide bridges was liquid chromatograph as described elsewhere carried out before separation of the cleaved (7, 15). BSA from the remaining uncleaved BSA, the CD measurements were carried out at 25°C bridges in the uncleaved BSA were also re- with a JASCO J-600 spectropolarimeter. In duced at the same time ( 12, 13). This reduced these measurements, a phosphate buffer of pH product gave four clear peaks when it was ap- 7.0 was used exclusively; the ionic strength of plied to a Superose 12 column (Pharmacia) the buffer was 0.014 for SDS solutions and connected to FPLC. Since one of the four 0.10 for urea and guanidine hydrochloride sopeaks corresponds to the disulfide bridge-re- lutions (6, 7, 12, 13, 15). The relative proJournal of Colloid and Interface Science, Vot. 144, No. 1, June t991

N-BROMOSUCCIN1MIDE-CLEAVED BSA portions of secondary structures were estimated by curve-fitting the CD spectrum (16), using the reference spectra of c~ helix, 13structure, and disordered form as determined by Chen et aI. (17). The simulation was carried out in the wavelength region 200-240 nm at 1-nm intervals. The simulated wavelength region was shifted to 210-240 nm with an increase in the concentrations of urea and guanidine hydrochloride (6, 12, 13, 15 ). RESULTS AND DISCUSSION The cleavage at Trp 134-Gly 135 and Trp 212Ser 213 in BSA was carried out with NBS. Cleaved BSA consists of three fragments which are linked by disulfide bridges. By reduction of these disulfide bridges, the three separated fragments were obtained. These fragments were determined to be Asp l-Trp 134, Gly 135_ Trp 212, and Ser213-Ala 582 within the sequence of the parent protein, on the basis of the analysis of amino acid residues at the N and C terminals of each. It has been reported that intact BSA binds about 200 dodecyl sulfate ions ( 18 ). We examined the effect of the cleavage at Tip 134Gly 135 and Trp212-Ser213 on the profile for binding of SDS to the protein. Figure l A shows the isotherm for binding of SDS to cleaved

47

BSA. As can be seen in Fig. 1A, the amount of bound SDS increased stepwise. The Saturated binding amount of about 240 m o l / m o l was apparently larger than the above 200 mol/ mol for intact BSA. SDS binding to the cleaved BSA in low surfactant concentrations was examined using the Scatchard plot (2) as shown in Fig. lB. The number of stoichiometric binding sites was estimated to be 14 as against 10 for the intact BSA (2, 19-21). Cleavage at the two sites affects both the total cooperative binding (2) and the stoichiometric binding. Secondary structures of cleaved BSA and the three isolated fragments were estimated by CD measurements. Although there is some uncertainty in absolute proportions of secondary structures determined by the method of curve-fitting CD spectra, the trend in their change can be easily and relatively accurately compared. The present curve-fitting was carfled out using the reference spectra of secondary structures determined by Chen et al. (17) in the same manner as in the case of the intact BSA (22). Their reference spectra have given a simulated CD spectrum of intact BSA, which is in excellent agreement with the experimentally obtained spectrum [see Fig. 1 in Ref. (22)]. Table I shows the o~-helical proportions of cleaved BSA and the fragments together

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TAKEDA AND WADA

48

TABLE I c~-Helical Proportion of Native BSA, Reduced BSA,a Cleaved BSA, and Isolated Fragments a-Helical proportion (%)

Native BSAb Cleaved BSAc BSA fragments with disulfide bridgesa AspS_Asp3o6 Arg193_Tyr368 Gln383 ile5~1 Reduced BSA~ RCAM-BSAe RCM-BSAf BSA fragments with no disulfide bridgec Aspl-Trp 134 Gly135 Trp212 Ser213_Ala582

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SDS

Guanidine

Urea

66 55

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7 (6.5 M) 10 (7.2 M)

15 (9 M) 15 (9 M)

57 60 51

44 (10 mM) 45 (10 mM) 46 (10 raM)

7 (6.4 M) 6 (6.4 M) 5 (6.4 M)

15 (9 M) 13 (9 M) 12 (9 M)

25 27

50 (10 raM) 42 (10 raM)

4 (6.5 M) 2 (6.4 M)

9 (7 M) 6 (8 M)

23 21 22

36 (10 raM) 36 (10 mM) 38 (10 mM)

6 (7.2 M) 6 (7.2 M) 3 (7.2 M)

9 (9 M) 4 (9 M) 8 (9 M)

a Disulfide bridge-reduced BSA. b From Refs, (12, 22). c This work. a From Ref. (6). e From Ref. (12) for BSA, disulfide bridges of which are reduced and then resultant sulfhydryl groups are blocked by iodoacetamide. fFrom Ref. (23) for BSA, disulfide bridges of which are reduced and then resultant sulfhydryl groups are blocked by iodoacetate. with those o f intact BSA ( 12, 22) and disulfide bridge-reduced BSA (12, 23). The a-helical proportion o f 66% for the intact BSA agrees very closely with that (22) estimated from the Brown model o f B S A ( 1 ). Cleavage at Trp 134_ Gly 135 and TrpZlZ-Ser213 in BSA caused an appreciable change in the secondary structure o f the protein: the proportion o f helical structure decreased to 5 5%. According to the Brown model o f B S A ( 1 ), the above two cleavage sites are located in the a-helical moieties in the second large loop in the first d o m a i n ( 1CX and 1CY according to Brown) and in the first large loop in the second d o m a i n ( 2 A X and 2 A Y also according to Brown). Each of these helical loops consists o f approximately 45 amino acid residues. Therefore, the aforementioned decrease in the proportion o f helical structure is considered to be due to disruption o f the a helices in the vicinity o f the cleavage sites by NBS. T h e approximately 10% decrease (66% Journal of Colloid and Interface Science, Vol. 144, No. I, June 1991

- 55%) corresponds to the disruption o f helices with about 60 residues ( = 5 82 × 0.1 ), that is, each cleavage of the two appears likely to cause the disruption o f a helical moiety with about 30 residues. As is seen in Table I, the three fragments had about 20% helical structure and about 10% [3 structure, although the lengths o f the fragm e n t polypeptides were very different from one another. This might be due to the fact that the p r i m a r y structure o f BSA consists o f the repetition of several similar sequences ( 1 ). The proportion o f helical structure in the present fragments with no disulfide bridge is clearly lower than those in the BSA fragments with disulfide bridges (also in Table I), the latter of which were prepared t h r o u g h pepsin digestion (6). Figure 2 shows secondary structural changes o f cleaved BSA (A) and isolated Ser213-Ala 582 (B) in SDS. The other two fragments also

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showed similar tendencies of secondary structural changes in SDS (not shown) to S e r 213Ala 582. The proportion of helical structure for cleaved BSA slightly decreased to 50% in SDS. It has been found that the helicity for intact BSA also decreases from 66% to 50% in SDS (22). If only the disruption of helical structure occurs in the same moieties in the cleaved BSA as in the intact protein, the helicity should be about 40% for the cleaved BSA. However, the proportion of helical structure was 50% for the cleaved BSA (Fig. 2A). On the other hand, the reduction of 17 disulfide bridges in BSA causes a drastic decrease in the proportion of helical structure to 25-27% ( 12, 23). The pro7" El

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FIG. 3. Relative proportions of a helix (~), B structure (fl), and disordered structure (D) of cleaved BSA (A) and Ser 213-Ala 582 ( B ) as a function of guanidine hydrochloride. Journal of Colloid and Interface Science,

Vol.

144,

No.

1, June

1991

50

TAKEDA AND WADA 7lg)

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noted that intact BSA (22), disulfide bridgereduced BSA (12), and the present cleaved BSA all attain the same extent of helical structure in SDS solutions above a certain concentration. In other words, these three types of BSA give almost the same CD spectrum in SDS solutions above a certain concentration. We have reported in a previous paper that SDS causes disruption of helical structures in the segments connecting domains of BSA (175197, 367-389) and in the segments connecting loops (62-74, 251-262, 446-458), and that the helices in the large loops are unchanged in SDS (6, 22). It therefore appears likely that the cleaved protein has two types of helicity, one of which is disrupted in SDS and the other of which is formed in SDS. It seems that, in SDS, the helical structures in these connecting segments in the cleaved BSA are disrupted, while the helical structures of the large loops, which were expected to be disrupted by the present NBS cleavage, are re-formed. Thus, the proportion of helical structure in the cleaved BSA in SDS would become the same magnitude as that of the intact BSA in the same surfactant solution. The secondary structural changes of the cleaved BSA and of Ser213-Ala 582in guanidine hydrochloride are shown in Figs. 3A and B, respectively. Figures 4A and B show the secondary structural changes of the cleaved BSA and of Ser 213_Ala582in urea, respectively. The other two fragments showed tendencies in secondary structural changes (not shown) Journal of Colloid and InterfaceScience, V o l .

144, No.

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1991

similar to those of Ser 213_Ala582 in guanidine and urea. The helical structure in the intact BSA resists urea and guanidine denaturation up to 4 and 1.5 M, respectively (12). In contrast, the helical structures in the cleaved BSA and the three fragments began to be disrupted at much lower concentrations of these denaturants. This indicates that cleavage of only two sites in 582 residues makes the entire protein structure unstable. At higher concentrations of urea and guanidine, the amounts of helical structure remaining in cleaved BSA are appreciably larger than those in the disulfide bridge-reduced BSA (12). This indicates that the effect of the present cleavage on the helical

1.0

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FIG. 5. Apparent extent ofconformational change (f, pp) estimated from changes in residue ellipticity at 222 nm (© for cleaved BSA and ZXfor intact BSA) and 270 nm ( e for cleaved BSA and • for intact BSA) as a function of guanidine hydrochloride. See text related to f~pp.

N-BROMOSUCCINIMIDE-CLEAVED BSA

structures is lower than that of the reduction of all disulfide bridges in the protein molecule. The stability of the cleaved BSA was examined also by the measurements of CD spectra in the near-ultraviolet region due to the behavior oftryptophan and tyrosine residues. Apparent extent of conformational change (f~pv) was estimated from changes in residue ellipticity at 270 nm as

A,,,, = {[el. -[e]}/{[e]~-

[el,:,}

where [O] N and [O] n are residue ellipticities in 0 and 7.2 M guanidine, respectively. For comparison, f~pp was also estimated from the residue ellipticity at 222 nm of the two BSAs. Figure 5 shows changes inf~pv for cleaved BSA and intact BSA at 222 and 270 nm as a function of guanidine hydrochloride. The CD intensity of the cleaved BSA at 270 nm changed at concentrations ofguanidine lower than that of the intact protein. The tertiary structure seems to become appreciably unstable by cleavage of the two sites. Similar tendencies were also observed in the urea denaturation (not shown). Although the CD intensities of the intact BSA at 222 and 270 nm began to change around 4 M urea, those of the cleaved BSA changed around 2 M urea. Interestingly, disruption of the tertiary structure (270 nm) appears to occur just before disruption of the secondary structure (222 nm) in the guanidine and urea denaturations.

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51

Secondary structural changes were also examined in the thermal denaturation. The intact BSA has a critical temperature range around 50°C where their conformational change becomes irreversible (24-28). The present cleaved BSA also showed complete recovery of its helical proportion in the thermal denaturation below 50°C. The proportion of helical structure in cleaved BSA is shown as a function of temperature up to 65°C in Fig. 6. When the temperature was raised, the proportion of helical structure gradually decreased to 33% at 65°C. On the contrary, when the temperature was lowered from 65 °C, the proportion gradually increased, but did not attain its initial value before raising the temperature (see Fig. 6 ). The proportion of helical structure in the intact BSA decreases to 44% at 65°C (28). The difference between the proportions of cleaved and intact BSA at 65°C might come from the initial disruption of the helical structures around the two cleaved sites as mentioned above. The recovery fraction (ratio of the proportion after cooling to the proportion before heating) was 0.8, similar to that for intact BSA, indicating that the present cleavage does not affect the thermal stability of the other helical moieties. Related to this thermal denaturation, it must be more useful to combine these CD data with a calorimetric study that gives direct information for thermally induced transitions. Cleavage of the two sites in the single polypeptide of BSA caused an appreciable disruption in the helicity of the protein. This effect remained through the urea and guanidine denaturations and also through the thermal denaturation. However, the disrupted moieties appeared to re-form the helical structures only in SDS. REFERENCES

0.3

0

20

40

60

TEMPERATURE/°C

FIG. 6. Dependence of c~-helical proportion of cleaved BSA on temperature. Circles and triangles designate the proportions determined with ascending temperature and descending temperature, respectively.

1. Brown, J. R., in "Albumin Structure, Function, and Uses" (V. M. Rosenoer, M. Oratz, and M. A. Rothschild, Eds.), pp. 27-51. Pergamon Press, Oxford, 1977. 2. Steinhardt, J., and Reynolds, J. A., in "Multiple Equilibria in Proteins," pp. 239-302. Academic Press, New York, 1969. Journal of Colloid and Interface Science. Vol. 144,No. 1, June 1991

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3. Jones, M. N., in "Biological Interfaces," pp. 101-130. Elsevier, Amsterdam, 1975. 4. Lapanje, S., in "Physicochemical Aspects of Protein Denaturation," pp. 156-179. Wiley-lnterscience, New York, 1978. 5. Reed, R. G., Feldhoff, R. C., Clute, O. L., and Peters, T., Jr., Biochemistry 14, 4578 (1975). 6. Takeda, K., Wada, A., Nishimura, T., Ueki, T., and Aoki, K., J. Colloid Interface Sci. 133, 497 (1989). 7. Wada, A., and Takeda, K., J. Colloid Interface Sci. 138, 277 (1990). 8. Patchornik, A, Lawson, W. B., and Witkop, B., J. Am. Chem. Soc. 80, 4747 (1958). 9. Patehornik, A., Lawson, W. B., and Witkop, B., J. Am. Chem. Soc. 80, 4748 (1958). 10. Ramachandran, L. K., and Witkop, B., J. Am. Chem. Soc. 81, 4028 (1959). 11. Ramachandran, L. K., and Witkop, B., in "Methods in Enzymology" (C. H. W. Hirs, Ed.), Vol. 11, p. 283. Academic Press, New York, 1967. 12. Takeda, K., Sasa, K., Kawamoto, K., Wada, A., and Aoki, K., J. Colloid Interface Sci. 124, 284 ( 1988). 13. Takeda, K., Sasa, K., Nagao, M., and Batra, P. P., Biochim. Biophys. Acta 957, 340 (1988). 14. Sober, H. A., and Harte, R. A. (Eds.), in "Handbook of Biochemistry (Selected Data for Molecular Biology)," 2nd ed., p. C-71. CRC Press, Cleveland, OH, 1973. 15. Takeda, K., Wada, A., Yamamoto, K., Hachiya, K.,

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17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27. 28.

and Batra, P. P., J. Colloid Interface Sci. 125, 307 (1988). Yang, J. T., Wu, C.-S. C., and Martinez, H. M., in "Methods in Enzymology," Vol. 130, p. 208. Academic Press, Orlando, FL, 1986. Chen, Y. H., Yang, J. T., and Chau, K. H., Biochemistry 13, 3350 (1974). Takeda, K., Miura, M., and Takagi, T., J. Colloid Interface Sci. 82, 38 ( 1981 ). Pallanseh, M. J., and Briggs, D. R., J. Am. Chem. Soc. 76, 1396 (1954). Reynolds, J. A., Herbert, S., Polet, H., and Steinhardt, J., Biochemistry 6, 937 (1967). Reynolds, J. A., Gallagher, J. P., and Steinhardt, J., Biochemistry 9, 1232 (1970). Takeda, K., Shigeta, M., and Aoki, K., J. Colloid Interface Sci. 117, 120 (1987). Batra, P. P., Sasa, K., Ueki, T., and Takeda, K., Int. J. Biochem. 21, 857 (1989). Imahori, K., Biochim. Biophys. Acta 37, 336 (1960). Lin, V. J. C., and Koenig, J. L., Biopolymers 15, 203 (1976). Wetzel, R., Becker, M., Behlke, J., Billwitz, H., B6hm, S., Ebert, B., Hamann, H., Krumbiegel, J., and Lassmann, G., Eur. J. Biochem. 104, 469 (1980). Anderle, G., and Mendelsohn, R., Biophys. J. 52, 69 (1987). Takeda, K., Wada, A., Yamamoto, K., Moriyama, Y., and Aoki, K., J. Protein Chem. 8, 653 (1989).