Toxicon. Vol. 16, pp. 453-460.
0041~0101/78/0901--0453502.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
CROTAMINE
CONFORMATION:
pH AND
EFFECT
OF
TEMPERATURE
O. G. HAMPE,* M. M. VOZ.~RI-HAMPE'~ and J. M. GONt~ALVES3~ *Departamento de Fisiologia, Farmacologia e Biofisica. URFGS. 90.000, Porto Alegre, RS. Brazil 1-Departamento de Bioquimica. URFGS. 90.000, Porto Alegre, RS. Brazil ~Instituto de Energia Atomica, Setor de Radiobiologia. Cidade Universit,'iria. S~.o Paulo, SP. Brazil (Accepted for publication 28 November 1977) O. G. HAMPE, M. M. VOZARI-HAMPEand J. M. GON(~ALVES.Crotamine conformation: effect of pH and temperature. Toxicon 16, 453460, 1978.--The pH-isomers of crotamine were characterized by spectropolarimetric titration. The neutral isomer (I) found at pH range between 4 and 8-5 corresponds to the native conformation. After heating at 80°C for 30 rain in 8 M urea, it retains full biological activity and the small spectral changes induced by heating are readily reversible. Compared to the neutral isomer, the acid isomer (II) occurs at pH values lower than 2-0 and has an unmasked group presumably a carboxyl, with a p K ' of 2.6. The basic isomer (III) has two groups with a pK' of 9'7 exposed. Also, the exposure of the Nterminal tyrosyl in II and the tyrosyl and two tryptophanyl residues in III are revealed by difference spectrophotometry. Both II and Ill represent partially pH-denatured conformations which undergo further structural unfolding upon heating. The unfolding AH ° for isomers II and lII were determined to be 8.2 and 6.1 kcal/mole, respectively. In addition, the spectral AH ° calculated from the spectrophotometric data gives information about the spatial displacement of residues (mainly tryptophanyl and tyrosyl) which are more pronounced in III than in II. INTRODUCTION
rattlesnake neurotoxin isolated from the Brazilian snake Crotalus durissus terrificus is a small, very basic protein (isoelectric point of 10.3 as determined by free electrophoresis at la = 0"1) and has no free-SH groups (GoN~ALVES, 1951; GON~ALVES, 1961). Despite the small number of amino acid residues (42) as compared with other snake neurotoxins (RYDEN et al., 1973; YANG, 1974), crotamine has a very specific toxic activity GONt~ALVES, 1956; MOUSSATCH~ et al., 1956). In a previous communication we described several ultraviolet Cotton effects in the O R D spectrum of crotamine recorded at pH 5.5 (HAMPE and GOYUALVES, 1976). The spectrum was not significantly changed in 8 M urea, even after heating at 60°C for 2 hr, indicating a stable tertiary structure of the toxin maintained by three disulphide bridges. The studies reported in this paper were undertaken in order to investigate the conformational stability of crotamine at different pH values and temperatures with special emphasis on exposure of residues. CROTAMINE, a
MATERIALS A N D METHODS Crotamine was purified from Crotalus durissus terrificus snake venom on a column of SP-Sephadex G-25 with a gradient of 0.5-3.0 M NaCI in 0.05 M ammonium formate buffer, pH 3.2, after gel filtration on Sephadex G-75 with 0.05 M ammonium formate buffer pH 3.4. In addition to the method described by HAMPEand GONfALVES(1976), for purity evaluation, amino acid composition was also determined and gave Lys 9, His 2, Arg 2, Asp 2, Ser 3, Glu 2, Pro 3, Gly 5, Cys 6, Met 1, Ile 1, Leu 1, Tyr 1, Phe 2, and Trp 2. Prior to the optical measurements the protein concentration was adjusted using the E 280 value of 11-1 × 103. M - 1 . cm-1 at neutral pH. The ultraviolet spectropolarimetric measurements were performed in a Fica Spectropol Spectropolarimeter I equipped with a thermostated cell holder. Common and difference spectrophotometric measurements were carried out in a Zeiss D M R 21 double beam spectrophotometer equipped with a thermostated 453
454
O. G. HAMPE, M. M. VOZ.&RI-HAMPE and J. M. GONCALVES
cell holder. For obtaining recordings the 1.000 and 0.437 cm (230 QS) suprasil pathway cells from Hellma were used. The biological activity of crotamine was assayed in mice by measuring the time required to paralysis of the hind legs since this depends on the toxin concentration (Go~ALVES, 1961). RESULTS Spectropolarimetric titration of crotamine at 293 nm is shown in Fig. 1. Three conformational isomers are characterized, each one in a distinct pH range. Isomer I (neutral [
[
I
I
I
I
150
I00
5O p/
0041~0101/78/0901--0453502.00/0
© Pergamon Press Ltd. 1978. Printed in Great Britain
CROTAMINE
CONFORMATION:
pH AND
EFFECT
OF
TEMPERATURE
O. G. HAMPE,* M. M. VOZ.~RI-HAMPE'~ and J. M. GONt~ALVES3~ *Departamento de Fisiologia, Farmacologia e Biofisica. URFGS. 90.000, Porto Alegre, RS. Brazil 1-Departamento de Bioquimica. URFGS. 90.000, Porto Alegre, RS. Brazil ~Instituto de Energia Atomica, Setor de Radiobiologia. Cidade Universit,'iria. S~.o Paulo, SP. Brazil (Accepted for publication 28 November 1977) O. G. HAMPE, M. M. VOZARI-HAMPEand J. M. GON(~ALVES.Crotamine conformation: effect of pH and temperature. Toxicon 16, 453460, 1978.--The pH-isomers of crotamine were characterized by spectropolarimetric titration. The neutral isomer (I) found at pH range between 4 and 8-5 corresponds to the native conformation. After heating at 80°C for 30 rain in 8 M urea, it retains full biological activity and the small spectral changes induced by heating are readily reversible. Compared to the neutral isomer, the acid isomer (II) occurs at pH values lower than 2-0 and has an unmasked group presumably a carboxyl, with a p K ' of 2.6. The basic isomer (III) has two groups with a pK' of 9'7 exposed. Also, the exposure of the Nterminal tyrosyl in II and the tyrosyl and two tryptophanyl residues in III are revealed by difference spectrophotometry. Both II and Ill represent partially pH-denatured conformations which undergo further structural unfolding upon heating. The unfolding AH ° for isomers II and lII were determined to be 8.2 and 6.1 kcal/mole, respectively. In addition, the spectral AH ° calculated from the spectrophotometric data gives information about the spatial displacement of residues (mainly tryptophanyl and tyrosyl) which are more pronounced in III than in II. INTRODUCTION
rattlesnake neurotoxin isolated from the Brazilian snake Crotalus durissus terrificus is a small, very basic protein (isoelectric point of 10.3 as determined by free electrophoresis at la = 0"1) and has no free-SH groups (GoN~ALVES, 1951; GON~ALVES, 1961). Despite the small number of amino acid residues (42) as compared with other snake neurotoxins (RYDEN et al., 1973; YANG, 1974), crotamine has a very specific toxic activity GONt~ALVES, 1956; MOUSSATCH~ et al., 1956). In a previous communication we described several ultraviolet Cotton effects in the O R D spectrum of crotamine recorded at pH 5.5 (HAMPE and GOYUALVES, 1976). The spectrum was not significantly changed in 8 M urea, even after heating at 60°C for 2 hr, indicating a stable tertiary structure of the toxin maintained by three disulphide bridges. The studies reported in this paper were undertaken in order to investigate the conformational stability of crotamine at different pH values and temperatures with special emphasis on exposure of residues. CROTAMINE, a
MATERIALS A N D METHODS Crotamine was purified from Crotalus durissus terrificus snake venom on a column of SP-Sephadex G-25 with a gradient of 0.5-3.0 M NaCI in 0.05 M ammonium formate buffer, pH 3.2, after gel filtration on Sephadex G-75 with 0.05 M ammonium formate buffer pH 3.4. In addition to the method described by HAMPEand GONfALVES(1976), for purity evaluation, amino acid composition was also determined and gave Lys 9, His 2, Arg 2, Asp 2, Ser 3, Glu 2, Pro 3, Gly 5, Cys 6, Met 1, Ile 1, Leu 1, Tyr 1, Phe 2, and Trp 2. Prior to the optical measurements the protein concentration was adjusted using the E 280 value of 11-1 × 103. M - 1 . cm-1 at neutral pH. The ultraviolet spectropolarimetric measurements were performed in a Fica Spectropol Spectropolarimeter I equipped with a thermostated cell holder. Common and difference spectrophotometric measurements were carried out in a Zeiss D M R 21 double beam spectrophotometer equipped with a thermostated 453
454
O. G. HAMPE, M. M. VOZ.&RI-HAMPE and J. M. GONCALVES
cell holder. For obtaining recordings the 1.000 and 0.437 cm (230 QS) suprasil pathway cells from Hellma were used. The biological activity of crotamine was assayed in mice by measuring the time required to paralysis of the hind legs since this depends on the toxin concentration (Go~ALVES, 1961). RESULTS Spectropolarimetric titration of crotamine at 293 nm is shown in Fig. 1. Three conformational isomers are characterized, each one in a distinct pH range. Isomer I (neutral [
[
I
I
I
I
150
I00
5O p/
