The role of acetic acid in the prevention of salt-induced aggregation of snake venom cardiotoxins.

Transcription

1 Vol. 44, No. 1, January 1998 Pages The role of acetic acid in the prevention of salt-induced aggregation of snake venom cardiotoxins. T. Sivaraman, T. K. S. Kumar, C. C. Huang and C. Yu*. Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan. Received September 22, 1997 Summary: Snake venom cardiotoxins (CTXs) exhibit a strong tendency to aggregate upon desalting and hence it is extremely difficult to prepare salt-free cardiotoxin(s). In the present study, we describe a new method for preparation of salt-free CTX based on dialysis against acetic acid. Based on experimental observation and the three dimensional solution structure of cardiotoxin analogue I]I from the Taiwan cobra (Naja naja atra), a molecular mechanism for the prevention of aggregation of cardiotoxins by acetic acid is discussed. In our opinion, the results obtained in the present study would pave way for elucidating the structural basis for the broad spectrum of biological activities exhibited by snake venom cardiotoxins. Snake venoms are a mixture of many different proteins of which the cardiotoxins (1-5) and the neurotoxins (6-8) are the most toxic. Cardiotoxins exhibit an wide array of biological properties such as depolarization and contraction of muscular cells, lysis of erythrocytes, selective killing of certain types of cancer cells and inhibition of protein kinase C activity. Due to the broad spectrum of biological properties exhibited by the snake venom cardiotoxins, extensive research is being carried out to understand the structure-function relationship in this class of toxins (9,10). Cardiotoxins are highly basic (PI>I 0.0), small molecular weight (6.5-7 Kda), all [3-sheet proteins cross linked by four disulfide bridges (11-16). The isolation of cardiotoxins involves fractionation on a cation exchanger using a linear salt gradient (17). Owing to the highly positive nature of cardiotoxins, they elute only at high salt concentrations (> 0.2 M) (4). Presence of salt is known to affect the function of snake venom cardiotoxins significantly (18). However, interestingly, *To whom all correspondence should be addressed cyu@chem.nthu.edu.tw /98/ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 desalting fractions of cardiotoxins using dialysis against water invariably results in aggregation of the protein. Repeated desalting by gel filtration is also found to be insufficient to eliminate the bound salt (17). In this context, in the present communication, we report an efficient procedure to desalt cardiotoxins. In addition, we also discuss the possible molecular mechanism(s) involved in the aggregation during dialysis. Materials and Methods Crude Taiwan Cobra (Naja naja atra) was purchased from Maimi Serpentarium- Laboratory, Maimi, USA. Acetic acid was obtained from E. Merck, Germany. 1- Anilino napthalene sulfonic acid was purchased from Sigma Chemical Co., St. Loins, USA. The low molecular weight cut-off (<2000 kda) dialysis bags were obtained from Spectrum Chemical Co., USA. All other chemicals used in this study were of high quality analytical grade. Fractionation of the crude venom: Fractionation of the crude venom (Naja naja atra) was carried out as per the procedure reported by Yang et al (17) with minor modifications. Fractionation was carded out on a SP-Sephadex C-25 cation-exchanger (Column size 2.8 x 35 cm) using M phosphate buffer (ph 7.5) with a linear NaC1 gradient ranging from M, at room temperature. Individual cardiotoxins fractions were pooled individually and lyophilized. Desalting: Desalting of cardiotoxin analogue (CTX I~ from Naja naja atra was carried out by two methods; namely by dialysis and gel-filtration chromatography. For desalting by dialysis, the lyophilised CTX HI fraction (40 mg obtained from the cation-exchange fractionation and containing the salt) was divided into two equal portions (by weight). Each portion was dissolved in 10 ml of water. These two portions were desalted by dialysis in two dialysis bags separately against water and 0.1M acetic acid. The extent of aggregation in each bag was monitored by drawing 2 ml aliquotes from the dialysis bag at appropriate time intervals and the absorbance of the solutions were measured at 600 nm using Beckman DU-50 spectrophotometer. It should be mentioned that after the spectroscopic measurement the protein solutions were replaced into the respective dialysis bags. Desalting of CTX HI fraction [containing salt(s)] by gel-filtration was carried out by loading the lyophilized powder disolved in 5 ml of water. Desalting was carried out on a Sephadex G-25 column using 0.1 M acetic acid as the eluent. The flow rate was set at 20 m//hr. The protein elution was monitored at 280 nm. The protein fractions were pooled and lyophilized and stored for further analysis. One-dimensional (1D) proton NMR: The 1D proton NMR spectra of the various CTX 1/I samples were recorded at 25~ on a Bruker DMX-600 spectrometer. All spectra were obtained with a spectral sweep width of 7507 Hz using sodium 3- (trirnethyl silyl) {2,2,3,3-1H} propionate (ch-tsp) as the internal standard. Circular Dichroism (CD): CD spectra of the various CTX HI were obtained on a Jasco J720 spectropolarimeter, using 0.2 mm and lmm pathlength cells. The instrument was standardized using ammonium camphor sulfonic acid. All spectra were corrected for background noise. The protein concentration was estimated based on the extinction coefficient of CTX HI at 280 nm. 30

3 Hemolytic activity: The hemolytic activity of CTX HI samples obtained upon desalting by dialysis (against acetic acid ) and gel filtration was tested on erythrocytes derived from human blood. Freshly collected human blood in sodium citrate buffer was washed in isotonic NaCI solution. The cell suspension was centrifuged to get a clear supematant. The washed cells were resuspended in the same buffer for hemolysis. Aliquots of the washed cells were then added and diluted to the desired CTX concentration to a final volume of 1 ml at 37 ~ After the desired incubation period, aliquots were pipetted out, centrifuged for 1 min at 3000 rpm in a microcentrifuge. The absorbance of the supematant was then measured at 540 nm for the determination of the lysis of human red blood cells. All measurements were carded out with appropriate controls. The percentage hemolytic activity of the CTX HI samples lysed by water is assumed as 100%. The protein concentration was not corrected for the concentration of the salt content. Structure Calculation: The three-dimensional solution structure of CTX HI was solved earlier by Bhaskaran et al (19) based on resonance assignments in the two dimensional 1H NMR spectra obtained on a 500 MHz Bruker DMX NMR spectrometer. For obtaining better spectral resolution, the two-dimensional homonuclear NMR experiments on CTX III were re-run on a 600 MHz Bruker DMX NMR spectrometer. We found that some of the resonance assignments reported earlier (19) were erroneous. Some of these errors in resonance assignments were also pointed out by Chiang et al (20). The three-dimensional solution structure was recalculated based on the corrected 1H-NMR assignments with a combination of the distance geometry and simulated annealing protocols using X-PLOR software (19, 21). Results and Discussion Fig. 1 shows the fractionation profile of the Taiwan cobra (Naja naja atra) on the cation - exchanger SP-Sephadex C-25. It could be seen that there are five cardiotoxin fractions eluting in the salt concentration range of 0.24 and 0.56 M. The cardiotoxin analogue m (CTXIII) is best characterized (in terms of the structure and biological properties) cardiotoxin isomer in the venom of the Taiwan Cobra (1,2,10-13). Hence, in the present communication, we report results obtained from experiments on CTX ITI. Desalting of the CTX m fraction (obtained on the cation exchanger) by dialysis against water shows that the solution turns increasingly turbid with time (Fig.2). This is exemplified by the steady increase in the 600 nm absorbance of the solution. The 600 nm absorbance increases around 3 folds in the time (5 days) for which the absorbance of the solution under dialysis was monitored. Interestingly, when the desalting of the CTX HI fraction was carded out by dialysis against 0.1M acetic acid, the turbidity of the solution dramatically decreased within a time span of 30 minutes (Fig. 2). This implies that acetic acid prevents the aggregation of the protein (CTX IN) which is observed upon dialysis against water. 31

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL CTX IV 0 Retention time (mins) 30 NTX,5-- = oo "-~ 0"3" u'rx Ill..0.56M 0 I/" L 0 I 500 I Eluent volume ( ml ) Figure 1: Elution profile of Naja naja atra venom on SP-Sephadex C-25 column. Column size: 2.8 x 35 era; Flow rate: 50 ml per min; Eluent: Phosphate buffer ( ph 7.5) with a gradient of 0.56 M NaC1; Protein Loaded: lgrn. Inset figure shows the two components obtained on a reversed phase (C18) HPLC column, from the unresolved fraction on the cation exchanger (CM- Sephadex). The eardiotoxin fractions in the elution profile given in Ref. 4 have been renamed. The cardiotoxin fraction are labelled in the order of their elution from the cation exchanger. Comparison of the CD spectra of CTX IJI samples obtained upon desalting by dialysis and gel filtration show interesting deviations (Fig. 3). It could be clearly seen that CTX HI sample which was desalted by dialysis against water (WCTX m) shows weak CD spectra in the far and near UV regions, implying that the protein is aggregated (Fig. 3). On the other hand, the CTX III sample dialyzed against 0.1 M acetic acid (ACTX lli) shows a CD spectra expected for the protein in the native state. The far UV region of the spectrum shows a negative CD extrema centered around 214 nm expected for an all 13-sheet protein such as CTX III (Fig. 3). The near UV-CD spectrum of the 32

5 ' 200",, ~ =" ~ lo0- O q ~ _ i] 30 Time of dialysis Figure 2: Percentage change in the absorbance at 600 nm (AA600) of the CTX HI fraction upon desalting by dialysis against (o) water and (-) 0.1M acetic acid. The units of the lower and upper x-axis scales are minutes and days, respectively. The direction of the arrows indicate the relevant x-axis. toxin sample dialyzed against acetic acid shows a positive ellipticity CD band centered at around 270 nm, indicating that the tertiary structure of the protein (CTX HI) is unperturbed due to dialysis against 0.1 M acetic acid (Fig. 3). The near UV CD spectra of CTX I/I sample desalted by gel-filtration (GCTX m) shows that 270 nm CD band (signifying tertiary structural interactions in the protein) is much weaker than that shown by ACTX HI. This implies that GCTX HI is partially denatured. The perturbation in the tertiary structural interactions in GCTX HI could be due to the binding of the salt ions which are not removed upon desalting by gel-filtration. It should be mentioned that CD spectral characteristics of GCTX HI does not alter much even upon repeated gelfiltration of the sample (GCTX HI) implying that this desalting procedure (gel filtration) is inefficient in completely removing the salt ions bound to the protein. The one-dimensional proton NMR spectra of WCTX 11I, ACTX HI and GCTX HI are shown in Figure 4. The 1D NMR spectra of WCTX HI shows line broadening in several resonance peaks. Line broadening is particularly evident in the aromatic region ( ppm) and in the peaks corresponding to the side-chain aliphafic protons (1-3 ppm) in the spectrum (Fig. 4). Line broadening of resonances in proton NMR is generally associated with restricted rotational motion due to aggregation of the protein molecules. The 1D N'MR spectrum of GCTX HI also shows line broadening in few of the resonances in the aromatic and aliphafic regions. Since no aggregation was observed in the 33

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 20.'... i,,,,...,t ~LI... F~rl,,, I +, i,,, ~ r ~ ~b -2020~, Wavelength (nm) lllillililii,,li+illlliilillillll +,,,i l"'4 Wavelength (rim) Figure 3: Far (A) and near (B) UV CD spectra of (a) WCTX m, (b) GCTX ]]I and (c) ACTX m. 34

7 BIOCHEMISTRY onc/ MOLECULAR BIOLOGY INTERNATIONAL b 'V ~jl I" _ m I I I I ppm Figure 4: One-dimensional proton NMR spectra of (a) WCTX III, (b) GCTX m and (c) ACTX III. spectrophotometric measurements in GCTX l]i, the observed line broadening could be due to partial unfolding of the protein resulting in the loss of some native tertiary structural interactions. Interestingly, the 1D NMR spectra ofactx m (Fig. 4) shows no line broadening effects in any region of the spectra implying that the native tertiary structural interactions are intact in ACTX III. Thus, the results of 1D NMR corroborates with the results obtain from CD spectroscopy. Fig. 5 shows that hemolytic activity of CTX m samples obtained by desalting separately using dialysis and gel-filtration methods. It could be noticed that hemolytic 35

8 12 10 r "~ 8- O 6 g ' I' I I i [ Time (rains) Figure 5: Time-dependent hemolytic activity of (*) ACTX [1I and (o) GCTX K[. The concentration of protein used in the hemolytic assay was 30 ~M. activity of cardiotoxin sample obtained upon dialysis against 0.1 M acetic acid (ACTX I~ is higher than the activity shown by the CTX m sample realized by desalting using the gel-filtration technique, at all time-points of activity measurement. The lower hemolytic activity of the CTX m sample obtained upon gel-filtration could be due to the presence of high amounts of salt. It is well known that gel-filtration, in many cases does not efficiently eliminate salt(s) from proteins (22). It would be of interest to understand as to how desalting by dialysis against acetic acid prevents protein aggregation. Comparison of the sequences of snake venom cardiotoxins reported to date (23) show that all cardiotoxins isoforms possess atleast 8 lysine and two arginine residues. To understand the role of the cationic residues, it is important to determine the distribution of these positively charged residues in the three dimensional structure of cardiotoxins. In this context, in the present study, the solution structure of CTX 111 was re-calculated* from corrected 1H NMR assignments. The three dimensional structure of CTX III in solution shows that the protein is a three finger shaped, all 13-sheet protein with three loops emerging from a globular head (Fig. 6). It can visualized that the ten cationic residues in the protein are (Fig. 7) spread on the surface of the molecule in dense clusters. Such cationic-clusters are potential binding "The atomic co-ordinates of the recalculated CTX III structure in solution has been deposited in the Brookheaven Protein Data Bank, Upton, New York. 36

9 Figure 6: A stereo-view of the best-fit superposition of the I2 NMR solution structures of CTX 111 as determined by dynamical simulated annealing calculations. Front view Rear view Figure 7: GRASP representation of the cationic clusters (dark patches) on the front and rear side of the CTX 11I molecule. sites for the anionic salt (CI', PO43"). Chakrabarty et al (24,25) analyzing the anionbinding regions in several proteins also demonstrated that the binding sites for the negatively charged ions are located near cationic-rich regions in proteins. The solution structure of CTX 1II reveals at least two dense cationic clusters on the concave side of the molecule (Fig. 7). It appears that cardiotoxins owing to their highly basic nature and presence of dense positively charged clusters can bind tightly to the negatively charged salt ions in the buffer such as C1- and PO43-. Tight and excessive binding of these anions could result in partial unfolding leading to greater exposure of the hydrophobic sites in the cardiotoxin molecule. The partial unfolding of the protein upon binding of the anions is evident in the CD and 1D ~ spectra of GCTX 1]/. The aggregation of the CTX III fraction (containing salt) observed upon dialysis against water is possibly as a result of intermolecular interactions among the exposed hydrophobic sites of the anion-induced 37

10 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL partially unfolded state of the protein (CTX 1]/). The lack of aggregation in CTX HI fraction dialysed against acetic acid could be due to the amphipathic character of the acid. Acetic acid, due to its methyl group could effectively bind to the exposed hydrophobic sites (on the anion-induced partially unfolded state) and thereby blocks aggregation by screening those aggregation-sensitive hydrophobic sites on the protein. In addition, the acetate anions (from acetic acid) can also act as counter-ions by binding to the unbalanced positive charges on CTX I/I, during dialysis. It should be mentioned that acetic acid (due to its amphipathic character) has been recently reported to help in the refolding of proteins (26). Although the conclusions drawn in the paper are based on the experiments on CTX ]]I from the Taiwan Cobra (Naja naja atra), we believe they should also be generally applicable to cardiotoxins isolated from other snake venom sources. This is the first time wherein a method to obtain salt-free cardiotoxin is described. Research on saltfree cardiotoxins should clear some of the existing controversy on the biological activity of snake venom cardiotoxins (1). Acknowledgment: We thank the National Science Council [NSC M ] and C. S. Tsou memorial medical research advancement foundation (VGHTH ) for the research grants. References 1. Kumar, T. K. S., Lee, C. S., and Yu, C. (1996) in Natural Toxins II (Tu, A. T and Singh, B. R Eds.) pp , Plenum press New York. 2. Yu, C., Bhaskaran, R. and Yang, C. C. (1994) J. Toxin. Toxicol. Rev, 13, Dufton, M. J. and Rider, R. C. (1983) Crit. Rev. Biochem., 14, Sivaraman, T., Kumar, T. K. S., Yang, P. W. and C. Yu. (1997) Toxicon (in press). 5. Bhaskaran, R., Yu, C. and Yang, C. C. (1994) J. Protein Chem. 13, Yu, C., Lee, C. S., Chaung, L. C., Shei, Y. R and Wang, C. Y. (1990) Eur. J. Biochem. 193, Yu, C., Bhaskaran, R., Chaung, L. C. and Yang, C. C. (1993) Biochemistry 32, Peng, S. S., Kumar. T. K. S., Jayaraman, G., Chang, C. C and Yu, C. (1997) J. Biol. Chem. 272, Chiou, S. H., Hung, C. C., Haung, H., Chen, S. T., Wang, K. T. and Yang C. C (1995) Biochem. Biophys. Res. Commun. 206, Kumar, T. K. S., Yang, P. W., Lin, S. H., Wu, C. V., Lei, B., Lo, s. J., Tu, S. C. and Yu, C. (1996) Biochem. Biophys. Res. Commun. 219, Kumar, T. K. S., Jayaraman, G., Lee, C. S., Sivaraman, T., Lin, W. Y and Yu, C. (1995) Biochem. Biophys. Res. Commun. 207, Sivaraman, T., Kumar, T. K. S. and Yu, C. (1996) Int. J. Biol. Macromol. 19,

11 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 13. Sivaraman, T., Kumar, T. K. S. Jayaraman, G., Han, C. C. and Yu, C. (1997) Biochem. J. 321, Jayaraman, G., Kumar, T. K. S., A~ar, A. I and Yu, C (1996) Biochem. Biophys. Res. Conunun. 222, A~ar, A. I., Kumar, T. K. S., Jayaraman, G., Samuel, D. and Yu, C. (1996) J. Biomol. Struc. Dyn., 14, Arunkumar, A. I., Kumar, T. K. S. and Yu, C. (1997) Biochim. Biophys. Acta Yang, C. C., King, K, and Sun, T. P. (1981) Toxicon 19, Fletcher, J. E and Jiang, M. S. (1993) Toxicon 31, Bhaskaran, R., Haung, C. C., Chang, D. K. and Yu, C. (1994) J. Mol. Biol., 235, 1291-I Chiang, C. M., Chang, S. L., Lin, H. J and Wu, W. G (1996) Biochemistry 35, Bhaskaran, R., Haung, C. C., Tsai, Y. C., Jayaraman, G., Chang, D. K. and Yu, C. (1994) J. Biol. Chem. 269, Fischer, L. (1980) In Laboratory Techniques in Biochemistry and Molecular Biology, Vol - 1, part - 11 (Work, T. S. and Burdon, R. H. Eds.) pp North-Holland press, Amesterdam. 23. Dufton, M. J., and Hider, R. C. in Snake Toxins (Harvey, A. LEd) pp , Pergman press Inc., New York. 24. Chakarabarti, P. (1994) Int. J. Pept. Prot.Res. 43, Chakarabarti, P. (1994) J. Mol. Biol. 239, Yang, P. W., Kumar, T. K. S., Jayaraman, G. and Yu, C. (1996) Biochem. Mol. Biol. Int. 38,