Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom

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1 Indian Journal of Biochemistry & Biophysics Vol. 42, April 2005, pp Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom Xianghu Liu 1, Xiaolong Xu, Jiexia Chen 1, Wenqi Liu 2 and Qingliang Liu 1* 1 Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, , P. R. China 2 Center for Physical Sciences, University of Science and Technology of China, Hefei, , P. R. China Received 24 May 2004; revised 28 February 2005 Acutolysin A, a protein isolated from the venom of Chinese Five-pace snake (Agkistrodon acutus) has shown marked hemorrhagic and proteolytic activities. In the present study, the effects of metal ions and an inhibitor EDTA on the fluorescence and function of autolysin A have been studied, by following fluorescence and activity measurements. Acutolysin A contains a Ca 2+ -binding site, which provides it with important structural stability, and a Zn 2+ -binding site, which is essential for its enzymatic activities. The removal of metal ions in acutolysin A by incubation with EDTA results in irreversible inhibition and complete denaturation, and a marked decrease in its fluorescence intensity. The fluorescence intensity of acutolysin A is also decreased in the presence of Cu 2+, Co 2+, Mn 2+ or Mg 2+, but does not change in the presence of Ca 2+, Cd 2+, or Tb 3+. Caseinolytic activity of acutolysin A is enhanced by Co 2+, Ca 2+ and Mg 2+, but is partly inhibited by Cu 2+, Mn 2+ and Tb 3+, and completely inhibited by Cd 2+. Both Zn 2+ and Co 2+ recover the loss of activity of the protein caused by Cd 2+. Keywords: Acutolysin A; fluorescence; metal ion; Agkistrodon acutus, zinc-metalloproteinases, EDTA, snake venom IPC Code: A61K35/58 Hemorrhagins, the biologically active proteins are found widely distributed in snake venoms and can cause the hemorrhage and damage of animal tissues 1,2. Most of these proteins have metal ions and some of them are zinc-metalloproteinases 3. Snake venom zincmetalloproteinases have similar active site and biological function with matrix metalloproteinases (MMP), and are responsible for degrading the proteins of extracellular matrix. Many of them are able to act on collagens in connective tissues. They belong to the MMP super-family and therefore can become the key targets in the drug design for anti-tumor and antiarthritis drugs 4. In addition, they are useful as convenient tool in studying the mechanisms of the ligand-receptor and cell-cell interactions 5. The venom of Chinese Five-pace snake (Agkistrodon acutus) contains several biologically active proteins that interfere with the coagulation process 6,7. Four hemorrhagins, namely acutolysins *To whom correspondence should be addressed Tel.: ; Fax: qliu@ustc.edu.cn Abbreviations: MMP, matrix metalloproteinases; holo-acutolysin A, Ca 2+ - and Zn 2+ -containing acutolysin A; apo-acutolysin A, Ca 2+ - and Zn 2+ -free acutolysin A; CA, caseinolytic activity; EDTA, ethylenediamine tetraacetic acid; PAGE, polyacrylamide gel electrophoresis. A-D showing hemorrhagic and proteolytic activities have been purified from the venom Recently, the structures of acutolysins A and C have been reported 12,13. Both acutolysins A and C have a molecular mass of 22 kda and contain a Zn 2+ binding in the active site and a Ca 2+ binding on the molecular surface. It has been demonstrated that Zn 2+ is essential for the activity of acutolysin A 13, but as to the function of Ca 2+, it is not clear. Chelators, such as Chelex-100 and EDTA 5 can remove the metal ions in native acutolysin A, like acutolysin D. The Ca 2+ - and Zn 2+ - containing acutolysin A is termed as holo-acutolysin A and the Ca 2+ - and Zn 2+ -free form as apo-acutolysin A. Fluorescence spectroscopy is an important analytical technique suitable for multicomponent analysis, due to its inherent sensitivity, selectivity and versatility 14,15. Acutolysin A contains both Trp and Tyr residues and the change of its conformation or microenvironment caused by the metal ions or inhibitor can be detected by fluorescence spectroscopy 13. In this paper, we report the effect of metal ions and an inhibitor EDTA on the conformation and function of acutolysin A using fluorescence and activity measurements. Materials and Methods Materials Lyophilized venom powder was provided by the TUN-XI Snakebite Institute (Anhui, P. R. China).

2 LIU et al.: EFFECT OF METAL IONS ON FLUORESCENCE AND ACTIVITY OF ACUTOLYSIN A 101 DEAE-Sephadex A-50 and Sephadex G-75 were obtained from Pharmacia (Uppsala, Sweden) and casein from the Shanghai Institute of Biochemistry (Shanghai, P. R. China). Chelex-100 was purchased from Bio-Rad Laboratories. All other reagents were of analytical reagent grade. Milli-Q purified water was used throughout. Acutolysin preparation Holo-acutolysin A was purified using a modified procedure of Gong et al 13. Briefly, crude venom (4 g) was dissolved in 15 ml of starting buffer (0.02 M Tris- HCl, ph 8.0) and a small amount of precipitate was removed by centrifugation. The clear supernatant was loaded onto a DEAE-Sephadex A-50 column ( cm), preequilibrated with starting buffer, and then eluted with a linear gradient from M NaCl in the same buffer. The eluted fractions were tested for their hemorrhagic and caseinolytic activities by the following assays. The fraction containing acutolysin A was pooled and further purified by gel filtration on a Sephadex G-75 column ( cm) equilibrated and eluted with 0.15 M NaCl. All the purification steps were done at about 4 C. The apo-acutolysin A was prepared by dialysis of purified holo-acutolysin A against a suspension of Chelex-100 (1 g/l; Bio-Rad) in 0.02 M Tris-HCl (ph 8.0). Caseinolytic activity of acutolysin A Caseinolytic activity (CA) was measured by the method described earlier 16. The reaction mixture containing 0.1 ml protease solution (1 mg/ml) and 2.0 ml casein solution (1%, w/v, 0.1 M Tris-HCl, ph 9.0) was incubated at 37 C for 30 min. Then 3.0 ml of trichloroacetic acid (5%) was added and the mixture further incubated at 40 C for 30 min, centrifuged at 4500 g for 15 min and the absorbance of the supernatant was recorded at 280 nm. One unit of caseinolytic activity was defined as the amount of enzyme, which induced a absorbance unit (AU) increasing in absorbance per min. Solutions of metal ions The solutions of Ca 2+, Zn 2+, Cu 2+, Cd 2+, Co 2+, Mn 2+ and Mg 2+ ions were prepared from their respective chlorides. Tb 4 O 7 was dissolved in conc. HCl by gentle heating to dryness, and then dissolved in Milli-Q super-pure water, respectively. The ph values of solutions were adjusted to 6.0 with HCl or NaOH. All metal ion solutions were standardized by titration with standard EDTA solution. Fluorescence measurements All fluorescence measurements were performed on a Shimadzu RF-5000 spectrofluorometer using a 1 cm quartz curette at an excitation wavelength of 280 nm at 25 C. The bandwidths for excitation and emission were both set to 5 nm. Each spectrum is the average of 3 consecutively acquired spectra. All spectra were corrected for buffer base-line fluorescence. Results Effect of ionic strength on CA of holo-acutolysin A Holo-acutolysin A (Ca 2+ - and Zn 2+ -containing acutolysin A) was purified by a two-step chromatography procedure of anion-exchange chromatography and gel permeation and its homogeneity was judged by PAGE and SDS-PAGE 13. Before analyzing the effect of metal ions on the CA of holo-acutolysin A, the effect of ionic strength on CA was determined. Since holo-acutolysin A has an optimum of ph 9.0 for CA (data not shown), ph 9.0 was used for analyzing the effects of ionic strength. As shown in Fig. 1, upon addition of NaCl, very little change of CA of holo-acutolysin A was observed. Effect of metal ions and EDTA on CA of holo-acutolysin A Holo-acutolysin A contains both Zn 2+ and Ca 2+, which can be replaced by other metal ions. The effect of metal ions on its CA of holo-acutolysin A was determined and the results are shown in Fig. 2. Addition of Co 2+, Ca 2+, or Mg 2+ ions increased the CA of holo-acultoysin A. However, while addition of Cu 2+, Mn 2+, Zn 2+ or Tb 3+ decreased the CA to different extents, addition of Cd 2+ resulted in the loss of the CA. In the presence of 0.1 mm Cd 2+, CA was restored by 80% upon the addition of Zn 2+ ion, and completely restored upon the addition of both Zn 2+ Fig. 1 Effect of ionic strength on the caseinolytic activity caseinolytic activity (CA) of holo-acutolysin A [The CA of holoacutolysin A at ph 9.0 in the presence of different concentrations of NaCl were determined as described in Materials and Methods. Each point represents the average of triplicate determination]

3 102 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, APRIL 2005 Fig. 2 Effect of metal ions on CA of holo-acutolysin A [The CA of holo-acutolysin A at ph 9.0 in the presence of Cu 2+, Cd 2+, Co 2+, Mg 2+, Mn 2+, Zn 2+, Ca 2+ and Tb 3+ were determined as described in Materials and Methods. The concentration of each metal ion is 1.0 mm. Each point represents the average of triplicate determination] and Ca 2+ (Fig. 3), however, the addition of Ca 2+ did not restore the CA. Interestingly, CA was also recovered after addition of Co 2+ (Fig. 4). EDTA, as an inhibitor of zinc metalloproteinase completely inhibited the CA (Fig. 5A). CA was not recovered after addition of Ca 2+, Zn 2+ or both Ca 2+ and Zn 2+ to apo-acutolysin A (Ca 2+ - and Zn 2+ -free acutolysin A). The fluorescence intensity of holoacutolysin A decreased after removal of Ca 2+ and Zn 2+ by addition of EDTA (Fig. 5 B), and was not restored after addition of Ca 2+, Zn 2+ or both Ca 2+ and Zn 2+. Effect of metal ions on fluorescence of holo-acutolysin A As acutolysin A contains both Trp and Tyr residues and belongs to type B proteins, its fluorescence emission by excitation at 280 nm should be dominated by Trp residues fluorescence, due to the intramolecular energy transfer from Tyr to Trp residues 17. To examine the metal ions-induced conformational change around Trp residues, fluorescence measurements were performed at an excitation wavelength of 280 nm. The wavelength λ max for Trp depends on its microenvironment. Specifically, a low polarity, hydrophobic microenvironment is characterized by λ max 331 nm, while for Trp in an aqueous phase λ max is nm 18. There are three Trp residues in acutolysin A 13, thus the average microenvironment of Trp residues can be assessed. The maximum excitation and emission of holo-acutolysin A are at 280 nm and 342 nm, respectively. The about 10 nm blue shift of λ max of holo-acutolysin A relative to that of Trp in an aqueous phase suggests that some Trp residues are located in hydrophobic environment. Fig. 3 Effect of Ca 2+ and Zn 2+ on the CA of Cd 2+ -acutolysin A [The CA of Cd 2+ -acutolysin A with 0.1 mm Cd 2+ in the presence of different concentrations of Ca 2+ or Zn 2+ were determined as described in Materials and Methods. Each point represents the average of triplicate determination] Fig. 4 Effect of Co 2+ on the CA of Cd 2+ -acutolysin A [The CA of Cd 2+ -acutolysin A in the presence of Cd 2+ and both Cd 2+ and Co 2+ were determined as described in Materials and Methods. The concentrations of Cd 2+ and Co 2+ are 0.1 mm and 1.0 mm, respectively. Each point represents the average of triplicate determination] The fluorescence spectra of holo-acutolysin A in the presence of different metal ions are shown in Fig. 6 A. No obvious changes for the maximum emission wavelength were observed after addition of different metal ions, suggesting that metal ions have no significant influence on the polarity of microenvironment of Trp residues. Addition of Cu 2+, Co 2+ and Mn 2+ markedly decreased the fluorescence intensity of the protein, while Mg 2+ slightly decreased and Ca 2+, Tb 3+ and Cd 2+ showed no effect on fluorescence intensity (Fig. 6 B).

4 LIU et al.: EFFECT OF METAL IONS ON FLUORESCENCE AND ACTIVITY OF ACUTOLYSIN A 103 Fig. 5 Effect of EDTA on the CA of holo-acutolysin A (A), and the effects of Ca 2+ and Zn 2+ on the fluorescence of apo-acutolysin A (B). Condition: 0.1 M Tris-HCl (ph 9.0), excitation 280 nm, final protein concentration 0.01 mg/ml, final metal ions concentration 1.0 mm] Fig. 6 The effect of metal ions on the fluorescence of holo-acutolysin A [Condition: 0.1 M Tris-HCl (ph 9.0), excitation 280 nm, final protein concentration 0.01 mg/ml, final metal ions concentration 0.2 mm. (A) Fluorescence spectra of holo-acutolysin A in the presence of different metal ions; and (B) Effect of metal ions on the fluorescence intensity at 340 nm of holo-acutolysin A] Discussion The present study was aimed to investigate the effects of metal ions on the activity and fluorescence of acutolysin A. Holo-acutolysin A loses all CA after removal of Ca 2+ and Zn 2+ or addition of Cd 2+. The loss of activity caused by Cd 2+ is restored, after addition of Zn 2+, but not with Ca 2+, suggesting that Zn 2+ is essential for its enzymatic activity. Ca 2+ is important for the structural integrity of the snake venom zincendopeptidase adamalysin II 19. In holo-acutolysin A, Ca 2+ probably plays a role similar to that in adamalysin II. This is based on the fact that the presence of Ca 2+ markedly enhances its CA. Holoacutolysin A loses its CA in the presence of 0.1 mm Cd 2+, and the activity is restored by 80% upon the addition of Zn 2+ ion, and completely restored upon the addition of both Zn 2+ and Ca 2+ suggesting that Ca 2+ provides the protein with important structural stability. To explore the metal ions-induced fluorescence spectroscopy change of acutolysin A, Trp residue fluorescence measurement was performed. Protein fluorescence intensity depends upon the degree of exposure of Trp side chain to the polar, aqueous solvent and upon its proximity to specific quenching groups, such as protonated carboxyl and imidazole groups and deprotonated εamino groups 20. Metal ions have different effects on the fluorescence intensity of the protein. Earlier, we reported that Co 2+, Ca 2+, Mg 2+, Cd 2+, Mn 2+, Zn 2+ and Tb 3+ have no direct collisional quenching effect on the fluorescence of tryptophan 21. The complex formation between protein and metal ions, which in turn perturbs the microenvironment around the relevant tryptophan residue(s) may be

5 104 INDIAN J. BIOCHEM. BIOPHYS., VOL. 42, APRIL 2005 responsible for the metal ions-induced fluorescence quenching of the protein. Metal ions have no significant influence on the polarity of microenvironment of Trp residues, as indicated by no obvious change for the maximum emission wavelength of holo-acutolysin A, after the addition of different metal ions. Ca 2+, Mg 2+ and Tb 3+ are hard acids and have similar chemical properties, therefore, Mg 2+ and Tb 3+ probably replace part or all of Ca 2+ in holo-acutolysin A. Similarly, Cu 2+, Co 2+, Cd 2+, and Mn 2+ are soft acids and have similar chemical properties, therefore, probably replace part or all of Zn 2+ in holo-acutolysin A. These substitutions have obvious quenching effects on its intrinsic fluorescence, suggesting that they affect the microenvironment of relevant Trp residue(s). The different fluorescence quenching of Mg 2+ and Tb 3+ indicates that they have different effects on the microenvironment of Trp residue(s), due to their different ionic radii and charge. As Mg 2+ and Ca 2+ have very similar chemical properties (elements of main group of II A), the addition of Mg 2+ also enhances the CA of holoacutolysin A. Tb 3+, as a trivalent ion has stronger ionic potential, thus its substitution for Ca 2+ possibly induces a large conformational change and decreases the CA of the protein. Similarly, as Cd 2+ and Zn 2+ have very similar chemical properties, Cd 2+ can completely replace Zn 2+ and thus results in the loss of all CA. However, since Cd 2+ is a toxic metal ion, it can t be used as a potential antidote for Agkistrodon acutus snakebite. Co 2+ shares with Zn 2+ the ability to accept unusual coordination environments. In addition, it can readily replace Zn 2+ to form functionally active derivatives of a large number of zinc enzymes 22. Co 2+ can replace Zn 2+ in hemorrhagic toxin e (obtained from Western diamondback rattlesnake), without affecting its activity 23. As for holo-acutolysin A, its CA increases 28.2% after addition of 1 mm Co 2+, suggesting that Co 2+ probably replaces Zn 2+, and the reconstitution of acutolysin A with Co 2+ results in the increase of its CA. This is further supported by the fact that Co 2+ can restore the loss of CA caused by Cd 2+. A large number of metallo-enzymes are known to contain zinc, a diamagnetic metal, whose complexes do not exhibit visible absorption spectra. These properties limit the suitability of zinc as a probe for the environment of the metal ion at the active site of such enzymes. In contrast, cobalt is a paramagnetic and gives rise to the visible spectra and therefore can be a very good environmental probe of Zn 2+ ion 24. Thus, the replacement of Zn 2+ by Co 2+ is very important and useful. In conclusion, Zn 2+ is essential for the enzymatic activities of holo-acutolysin A, while Ca 2+ is important to stabilize its conformation. EDTA completely inhibits its activity and markedly decreases its fluorescence intensity irreversibly. Caseinolytic activity of holoacutolysin A is enhanced by Co 2+, Ca 2+ or Mg 2+, but is partly inhibited by Cu 2+, Mn 2+ or Tb 3+, and completely inhibited by Cd 2+. Both Zn 2+ and Co 2+ recover the loss of activity caused by Cd 2+, and Ca 2+ can help Zn 2+ to recover the activity. The fluorescence intensity of holoacutolysin A decreases in the presence of Cu 2+, Co 2+, Mn 2+ or Mg 2+, and does not change in the presence of Ca 2+, Cd 2+, or Tb 3+. MMP super-family is involved in various connective-tissue diseases, such as arthritis and breast cancer 4. The present study may prove useful for understanding the biochemical basis of acutolysin A, as a MMP, and also for designing the anti-tumour and anti-arthritic drugs. Zn 2+, Ca 2+, Co 2+, Mg 2+, Cu 2+, Mn 2+, and EDTA may be used as the regulators for the activity of acutolysin A in its application in drug design. Also, Co 2+ and Tb 3+ may be useful as the paramagnetic probes of Zn 2+ and Ca 2+, respectively for studying the structures of the metal ion sites in holoacutolysin A by electron paramagnetic resonance spectroscopy. Acknowledgement The work was supported by grants from the National Natural Science Foundation of China (No , X. L. Xu) and the Anhui Provincial Natural Science Foundation (No , X. L. Xu). References 1 Kamiguti A S, Hay C R M, Theakston R D G & Zuzel M (1996) Toxicon 34, Bjarnason J B & Fox J W (1995) Methods Enzymol 248, Bjarnason J B & Fox J W (1994) Phar Ther 62, Blundell T L (1994) Nature Struct. Biol 1, Xu X L, Liu X H, Wu B, Liu Y, Liu W Q, Xie Y S & Liu Q L (2004) Biopolymers 74, Xu X L, Liu Q L & Xie Y S (2002) Biochemistry 41, Xu X L, Liu Q L, Yu H M & Xie Y S (2002) Protein Sci 11, Xu X, Wang C, Liu J & Lu Z (1981) Toxicon 19, Chen Z L, Liu Q L, Wang S Y, Xu X L & Yu H M (1995) Spectrochim Acta A 55, Zhu Z, Gong W, Niu L, Teng M & He H (1996) Acta Crystallogr D 52,

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