Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography

Binding of copper and zinc to three cyanobacterial microcystins quantified by differential pulse polarography

Pergamon PII: S0043-1354(97)00033-X IVat. Res. Vol. 31, No. 7, pp. 1679-1686, 1997 © 1997 ElsevierScienceLtd. All rights reserved Printed in Great Br...

702KB Sizes 0 Downloads 3 Views

Pergamon PII: S0043-1354(97)00033-X

IVat. Res. Vol. 31, No. 7, pp. 1679-1686, 1997 © 1997 ElsevierScienceLtd. All rights reserved Printed in Great Britain 0043-1354/97 $17.00 + 0,00

BINDING OF COPPER AND ZINC TO THREE CYANOBACTERIAL MICROCYSTINS QUANTIFIED BY DIFFERENTIAL PULSE POLAROGRAPHY A M A N D A V. HUMBLE, G E O F F R E Y M. G A D D and G E O F F R E Y A. CODD* Departraent of Biological Sciences, University of Dundee, Dundee DD1 4HN, Scotland, UK (First received November 1996; accepted in revised form February 1997)

Abstraet--Microcystins are a family of potently hepatotoxic heptapeptides which have been detected in a wide range of cyanobacteria. The novel application of polarography in the analysis of these cyclic peptides ha,,; demonstrated binding of copper and zinc to microcystins in the aqueous-phase at environmentally-relevant pH values. This electroanalytical technique measured the shift in reduction potential and decrease in height of metal polarogram peaks as a function of increasing microcystin concentration. The successive formation of intermediate complexes was investigated and the strength of coordination~was evaluated in terms of a formation constant (K0 for each metal/microcystin combination. The effects ~f a single amino acid substitution on the formation constants of the heptapeptide was investigated with microcystin-LR, microcystin-LW and microcystin-LF. The calculated formation constants (log Kt) indicated that all three microcystins are intermediate strength metal ligands and that substitution of arginine with either tryptophan (microcystin-LW) or phenylalanine (microcystin-LF) appeared to have no discernible effect on the overall binding capacity. Reducing the pH of the supporting electrolyte strongly influenced complexation of microcystin-LR with copper, indicating that at pH 5.5 only a weak labile complex was formed. Complexation between microcystin-LR and zinc, however, was not influenced as strongly by pH. © 1997 Elsevier Science Ltd Key words--cyanobacteria, blue-green algae, microcystins, copper, zinc, formation constants, differential pulse polarography

INTRODUCTION Cyanobacterial rnicrocystin peptides comprise a family of chemically unique secondary metabolites which have receiw:d considerable attention owing to their action as potent hepatotoxins (Carmichael, 1994; Bell and Codd, 1994) but which, as yet, have no confirmed natural function. Five of the amino acids within these cyclic heptapeptides are highly conserved and are largely invariant. Two L-amino acids can be substituted and are used to name a particular microcystin. To date, over 50 microcystins have been characterised (Rinehart et al., 1994), the most widely studied being microcystin-LR (Fig. 1) which has leucine and arginine as the variable amino acids (Carmichael et al., 1988). Any theory attempting to explain the presence and role of microcystins must account for their remarkably conserved structure, whilst addressing the significance of this wide array of structurally-related toxins. Examination of the microcystin peptide with respect to determining possible structure/function relationships reveals that the cyclic peptide has numerous functional groups, such as = O and - N, which could act as potential metal coordination sites. Comparisons can be made with other cyclic peptides of *Corresponding author

bacterial origin, e.g. the cyclic decapeptide antibiotics (ferrocins) produced by Pseudomonas fluorescens YK-310, which bind iron in the central cavity of the cyclic structure, coordinating via O: groups (Katayama et al., 1993). The expression of microcystins can be influenced by environmental factors (Watanabe et al., 1992) including metal cation nutrition (Lukac and Aegerter, 1993; Campbell, 1994; Utkilen and Gjolme, 1995) which is of particular interest since metals such as copper and iron are used extensively in the control of cyanobacterial and algal blooms (e.g. Horne and Goldman, 1994). Copper, although an essential micronutrient, can be highly toxic at higher concentrations and has been widely employed as an algicide in the treatment of cyanobacterial and algal blooms. Upon treatment of a bloom, microcystins are released into the waterbody (Kenefick et al., 1993) and are hence available for complexation. Such interactions may have implications for the physicochemical form of copper and the biological availability and toxicity of complexed microcystins. Polarography has been used to study the complexing ability of extracellular organic compounds produced by Microcystis aeruginosa to bind copper (Ogiwara and Kodaira, 1989). The complexing capacities of the exudates produced at each growth phase were found to vary, with low molecular weight material ( < 103 Da) produced by



A.V. Humble et al.

stationary and death phase cultures having the highest stability constants. Microcystins (approximately 103Da) are largely retained within the producer-cells and are not released in appreciable quantities until cell lysis (Codd et al., 1989). Cyanobacteria and algae also produce metal-binding proteins which internally sequester and detoxify high concentrations of metals within the cell (Birch and Bachofen, 1990). Phytochelatins, for example, are small metal-binding peptides synthesised by algae in response to high concentrations of metals, e.g. Cd 2+ and Cu 2+ (Ahner and Morel, 1995). As an alternative to the direct algicidal effects of copper, ferric sulphate is used as a phosphate-stripper in eutrophic waterbodies to reduce cyanobacterial growth. Utkilen and Gjolme (1995) reported that excess iron caused an increase in microcystin levels in a laboratory culture of M . aeruginosa, a finding of particular interest due to its implications for iron-dosing policy. These authors have proposed that microcystin production is regulated by iron in a mechanism requiring binding between iron atoms and microcystin peptides. Laulhere et al. (1992) reported the first isolation of an iron storage protein, bacterioferritin, in a cyanobacterium, namely Synechocystis 6803. However, only 1% of intraceUular iron was sequestered in this form, whilst the remainder was associated with an, as yet, undefined low molecular weight peptide pool. The possible metal-binding potential of microcystins was investigated in the present study using the electroanalytical technique of polarography (Riley and Watson, 1987). In addition to the quantification of metal ion mixtures, polarographic methods are

now widely used in the analysis of organic compounds including fungal metabolites (Gadd and Edwards, 1986), antibiotics (Beard et al., 1992) and macromolecules of environmental origin such as humic and fulvic acids (Filella et al., 1990). This work describes the interaction between three microcystins and copper and zinc at environmentally-relevant pH values and estimates the strength of binding in relation to formation constants calculated from polarographic data. Copper was selected for investigation due to its direct relevance to the ecophysiology of cyanobacteria and was compared with zinc, a metal also required as a micronutrient in cyanobacterial growth. The influence of amino acid substitution in the common cyclic peptide structure was evaluated by comparison of the complexation characteristics and formation constants of microcystin-LR (MCLR), microcystin-LW (MC-LW) and microcystin-LF (MC-LF). In MC-LW and MC-LF, a strongly basic amino acid, arginine, is replaced by two non-polar amino acids, tryptophan and phenylalanine respectively (Craig et al., 1993; Azevedo et al., 1994; Lawton et al., 1994). Amino, carboxyl, nitrogen and oxygen groups within the microcystin molecule are potentially available for characteristic coordination bonding with metallic ions (Crist et al., 1981). Such bond formation can be accompanied by displacement of protons, which is partially dependent on the extent of protonation as determined by pH. This pH effect has been reported by Ruiz et al. (1995) who, using differential pulse polarography (DPP), demonstrated that the electrochemical behaviour of the metal-binding proteins, metallothioneins, from rabbit liver and






_.'~,H HaC Adda

~H= O



Ala H=C'/~ O


I ~' CH= HI HO~'1'. NH H

H...~CH= ~" o





HN o



¢ OH

I //Cx HN NH=

Arg Fig. 1. Structure of microcystin-LR. Abbreviations: Ala, alanine; Leu, leucine; Masp, methylaspartic acid; Arg, arginine; Mdha, N-methyldehydoalanine; Adda,/Lamino acid; 3-amino-9-methoxy-2-6, 8-trimethyl10-phenyldeca-4, 6-dienoic acid; Glu, glutamic acid (see Rinehart eta/., 1994).

Binding of copper and zinc to microcystins horse spleen was dependent on the p H of the solution. The effect of pH on copper and zinc binding by M C - L R was therefore also investigated by determination of formation constants at different pH values in order to elucidate the binding mechanisms involved.


Organism and growth conditions Microcystis aeruginosa PCC 7813 was grown photoautotrophically and axenically in 18 1 batch volumes of BG-l 1 medium containing nitrate (Stanier et al., 1971). Cultures were maintained at 2Y'C under continuous illumination by cool white fluorescent tubes, providing a surface irradiance of about 20 #E.m-2.s -~. Vessels were sparged with sterile air throughout the growth period until cultures reached late log phase (approximately 28 d). Cells were then harvested by centrifugation at 13,200 x g using a continuous flow centrifuge (Sharpies Centrifuge Ltd). Purification of microcystins Cell pellets were lyophilised and extracted in 70% (v/v) HPLC-grade methanol (BDH), acidified with 0.1% (v/v) trifluoroacetic acid (TFA) according to a protocol modified from Lawton et al. 1994). Each rnicrocystin variant was purified by semi-preparative high performance liquid chromatography (HPLC) with fraction collection of the column eluate. All equipment was obtained from Waters Ltd., Watford, U.K. ~Lndconsisted of two solvent delivery pumps used in conjut~etion with a WISP auto-sampler. Detection was by a variable wavelength detector set at 238 nm. A mobile phase of ammonium acetate 1 g/l and acetonitrile (Rathburn, Walkerburn, U.K.) was used with a Waters RCM prep column (Preparative Nova-Pak HR C~8; 6 #m particle size; 25 mm i.d. x 100 mm). This system was run isocratically at 30% (v/v) acetonitrile for 30 min at a flow rate of 1 ml/min. Such conditions were found to be optimal for resolution of the closely-eluting variants, MC-LW and MC-LF. Following extraction and purification, the purity of microcystin MC-LR, MC-LW and MC-LF was determined by analytical HPLC with diode array detection (DAD). Equipment consisted of a Waters solvent delivery system combined with a diode array detector and a WISP auto-sampler. Samples were separated on a C,s #Bondapak column (3.9 i.d. x 300 mm) using a mobile phase consisting of Milli-Q water (Millipore) and acetonitrile both containing 0.1% (v/v) TFA (Fisons). An acetonitrile gradient starting at 30% (v/v) was increased to 35% over the first 10 rnin followed by an increase up to 70% over the subsequent 30 min. Column temperature was maintained at 40°C using a Waters temperature-control module. Microcystins with a level of purity greater than 95% were subjected to polarographic analysis. Polarography Differential pulse polarography was performed using a Metrohm 663 VA stand incorporating a multi-mode electrode set to the dropping mercury electrode (DME) facility. Analysis was controlled by General Purpose Electrochemical System (GPES) software, version 3 (Eco Chemie). The electrtxie vessel contained 10 ml of 0.1 M KCI in Milli-Q water, buffered with either 10 mM PIPES or MES at the required pH, to which the desired metal compound (ZnSO4.7H20, o r CuSO4.5H20) was added from a freshly-prepared 5raM stock solution to give a final concentration of 100 #M. Initial and final voltage settings were +0.12 to -0.25 V and +0.60 to - 1.30 V for copper and zinc respectively. Glassware was thoroughly rinsed with 1 M nitric acid followed by multiple rinses with Milli-Q water and all chemicals were of reagent-grade purity.


10 mM stock solutions of HPLC-purified microcystins were prepared in methanol and added sequentially to the reaction vessel in 10#1 aliquots to give the required final concentration. Solutions were deoxygenated in situ for 5 min before polarograms were recorded, with each polarogram being obtained from four consecutive voltage sweeps, the summation of which was automatically statistically smoothed to enhance the signal to noise ratio of experimental sets of data. Computer analysis then provided values for the evolution potential and current potential of the smoothed peak. Throughout the experiments a constant temperature of 25°C was maintained by partial submersion of the electrode vessel in a water bath and all solutions were allowed to equilibrate at this temperature before addition to the vessel. 100 #1 aliquots of methanol were sequentially added to metal solutions in a separate experiment in order to ascertain that this solvent, used to reconstitute extracted microcystins, had no chelation effect or did not interfere with the analysis. RESULTS Addition of increasing amounts of microcystin-LR to either a 10-4M CuSOa.5H20 or ZnSO4.7H20 solution resulted in a concentration-dependent decrease in the polarogram peak signal which corresponds to the metal present. Decrease in metal peak height was associated with a shift in the evolution potential (Fig. 2a, b), suggesting metal-peptide interaction. Plotting the evolution potentials obtained after each addition against the logarithmic value of M C - L R concentration produced a curved line (data not shown). This was indicative of the formation of consecutive complexes (Lingane, 1941) and therefore the method of Deford and Hume (1951), modified by Heath and Hefter (1977), was applied in order to calculate the formation constant (Kn) for each complex. Two F,(X) functions were derived from each set of data, corresponding to two consecutive complexes. A plot of calculated F0(X) values against M C - L R concentration produced a curved line (Fig. 3a, b). The /71(70 values subsequently calculated from these F0(X) values were plotted on the same graph, giving a straight line which was extrapolated by regression analysis to zero M C - L R concentration and the F~(X) value at the y-intercept was assigned the value for K~. Substitution of this value and calculation of F2(X) values produced a straight line parallel to the x-axis which, on extrapolation, gave a value for/('2. The formation constants for sets of data from triplicate experiments were calculated in this way and the mean values for the c o p p e r / M C - L R and the z i n c / M C - L R system are presented in Table 1. M C - L W and M C - L F appeared to have the same polarographic behaviour as M C - L R , in that the addition of M C - L W or M C - L F aliquots to solutions of copper or zinc caused a reduction in metal peak height accompanied by a shift in evolution potential to more negative values. These effects were concentration-dependent and data were processed according to the method of Deford and Hume (1951) as previously described for M C - L R . Graphical derivatisation of calculated F,,(X) functions provided formation constant values for two


A.V. Humble et al.

(a) -3.00.g',



:j 0.I0

~ /




shift in evolution potential was observed, confirming that the changes in the polarograms with microcystins were not due to the methanol content of the aliquots and that methanol did not interfere with electrochemical processes during polarography. The stability of many metal complexes is dependent on pH (Riley and Watson, 1987). The effect of pH on the complexation characteristics of MC-LR with copper and zinc was investigated in buffered metal solutions at pH 5.5, 6.5 and 7.5 and the formation constants for each metal/MC-LR/pH combination were calculated (Table 2). As previously demonstrated, MC-LR produced a well-defined complexation effect with copper at pH 7.5, in that the Cu 2+ ion peak amplitude was markedly reduced upon addition of MC-LR and that this reduction coincided with a shift in evolution potential (Fig. 2a). Lowering

-].6o -2.b0


Evolution potential (V)

18. (a) 16. 14.




~-" -].20- (b)


o~u 2.5~ Micr~y~in-LR coneentrafion 0B~I)

i l / ~ " 7.5ttM

80 -



60. -0.80

-1.00 -1.20 -1.40 Evolution potential (V) 40.

Fig. 2. Polarograms of (a) copper-binding (b) zinc-binding by microcystin-LR. Analysis was performed at 25°C in 0.1 M KCI containing either CuSO~.5H20 or ZnSO4.7H20, added to a final concentration of 10-4 M and buffered at pH 7.5 using PIPES. Typical traces are shown from one of three experiments. Final microcystin-LR concentrations are indicated.



ol 5

consecutive complexes and the calculated means from three experiments are shown in Table 1. The microcystin variants added to the polarographic system were resuspended in HPLC-grade methanol and, as a control, 100 #i aliquots of methanol were added to metal ion solutions (polarograms not shown). In each case, no decrease in peak current or

ID " 1'5 " 2'0 " 2"5

Microcy~Cin-LR conc~Wation (f~M) Fig. 3. Derived F.(X) functions of (a) coppcr/microcystinLR (b) zinc/microcystin-LR. Analysis was performed at 25°C in 0.1 M KCI containing either CuSO4.5H20 or ZnSO4.7H20, added to a final concentration of 10-4 M, and buffered at pH7.5 using PIPES. F,(X) values are represented by; (O)=F0(X), ( m ) - - F , ( X ) × 105 and

([3) = F2(X) x I0 '°.

Binding of copper and zinc to microcystins


Table I. Peptide-metal formation constants for microcystin variants. Analysis was performed at 25°Cin 0.1 M KCI containingeither CuSO4.5H20or ZnSO4.7H.,O,added to a finalconcentrationof 10-4M and bufferedat pH 7.5 usingPIPES Co,2+ log formation constant (K,) (/(2)

Ligand microcystin-LR mi,:rocystin-LW microcystin-LF

5.13 4- 0.43 4.97 4- 0.21 5.43 _+ 0.52

9.83 _+ 0.92 10.93 + 0.39 10.47 +_ 0.61

the electrolyte/meta![ solution to pH 6.5 and subsequent titration with MC-LR resulted in a peak height decrease but with a negligible associated shift in evolution potential, indicating the formation of weaker, labile complexes. The lack of appreciable evolution potential shift meant that no information about the complex stability could be deduced over the MC-LR concentration range used. In the presence of a large excess of ligand it is possible that more revealing potential shift data could be obtained. Further lowering of the pH to 5.5 resulted in minimal peak height decrease with no evolution potential shift, indicating that an even weaker complex had formed. Functional groups available for coordination at pH 7.5 are increasingly protonated at lower pH values so that the metal ion must displace H ÷ ions from the toxin molecule before complexation. This may explain the pH-dependent gradation of complexation behaviour. The pH effect on metal/MC-LR interactions is better visualised by plotting the normalised peak current against ligand concentration. The normalised peak current (0) is defined as the ratio of the peak current in the presence of a ligand to that measured in the absence of a ligand (Diaz-Cruz et al., 1'992). Figure 4a shows that of all pH values tested, the greatest decrease in peak current occurred o~er the lower end of the MC-LR concentration range as reflected by the rapid decrease in 0. Thereafter this effect declined gradually with almost complete quenching of the Cu 2+ ion peak at pH 7.5 at a final MC-LR concentration of 30 pM. Over this range it can be seen that 0 values only decreased slightly at pH5.5, but the gradual downward slope does, however, imply that further, excessive additions would eventually result in complete quenching of the ion peak. It can therefore be concluded that the interaction of MC-LR with Cu 2+ is pH-dependent. A similar investigation with MC-LR and 10-4 M solutions of ZnSO,7H20 revealed that the voltamTable 2. Peptide-metal formation constants for microcystin-LR. Analysis was performed at 25°C in 0.1 M KCI containing either CuSO4.SH20 or ZnSO4.7H20, added to a final concentration of 10 -4 M and buffered with either PIPES or MES

pH 5.5 6.5

Cu::+ log formation constant (K 0 (K2) ND ND


Zn2+ log formation constant (Kn) (1(2) 5.39 :t: 0.42 5.08 + 0.01

11.33 __ 0.38 11.09 4- 0.08

ND ~ non determinable.Changesin polarogramevolutionpotential were insufficientfor calculationof formationconstants.

Zn 2+ log formation constant (Kt) (K2) 5.15 4- 0.17 5.31 4- 0.23 5.04 -4- 0.24

10.70 4- 0.33 10.76 4- 0.43 10.40 4- 0.15

metric response was essentially constant over the pH range tested, as determined by comparison of the mean Kt and /(2 formation constant values derived from metal ion peak parameter data (Table 2). Polarograms obtained at pH 5.5 showed that an equally strong chelation effect can be obtained even at this lower pH value and both reduction in peak current and shift in evolution potential were clearly observed. When the peak parameter data obtained for these experiments are expressed in terms of normalised peak current and then plotted against the

~ (a)


0.6 "~ 0.4



~ 0.2 0-00

5 " lb' l~'io'



Microcystin-LR concentration (IxlVl)

(b) 1.01

0.8 0.6 0.4

0.2 0.0 o • ~ " ,i " 8 " ~ ' l b ' f 2 ' f 4 ' f 6 ' Microcystin-LR concentration (0M) Fig. 4. Normalised peak current (0) as a function of microcystin-LR concentration in (a) 10-4 M CuSO4.5H.,O (b) 10-4 M ZnSO4.7H20. Results obtained at pH 5.5 (Q), pH 6.5 (m) and pH 7.5 (l'q).


A . V . Humble et al.

ligand concentration (Fig. 4b), it can be seen that pH influenced zinc/MC-LR interaction. This effect is not evident in the derived formation constants because, in their calculation, the mathematical emphasis is placed on the observed shift in evolution potential from the half-wave potential of the uncomplexed metal ion, with the peak current data being incorporated as a log term only (Deford and Hume, 1951). Hence, peak current data have limited influence on the final calculated formation constants so that variations were not readily apparent. From Fig. 4b it can be seen that the zinc ion peak was substantially quenched upon the addition of 15#M MC-LR. Surprisingly, a greater decrease in 0 was found at pH 5.5 than at pH 7.5. This is difficult to explain since greater protonation of the microcystin molecule at the lower pH should have reduced complexation. The pH effect on zinc/MC-LR complexation was not substantial, as confirmed by similarity in formation constants, but was still evident.


These studies have obtained evidence for microcystin-metal cation interactions in the aqueous phase. Polarography has been shown to be a non-destructive analytical technique which has clearly demonstrated complexation between microcystins and metal cations and has allowed the successive formation of intermediate complexes to be studied and the strength of this coordination to be evaluated. Control experiments confirmed that the changes in metal ion peak parameters were specifically due to the addition of toxins to the system and these changes were used to calculate the formation constants for each mierocystin in either copper or zinc solutions. Experiments performed at the environmentally-relevant pH of 7.5 ensured that the ligands were de-protonated, with the dissociation of protons from functional groups creating new binding sites and thus providing optimal conditions for complexation. Solutions were buffered in order to maintain this optimal state and, under these defined conditions three microcystins, MC-LR, MC-LW and MC-LF produced similar complexation effects with copper and zinc. The derivation of two F~(X) functions from the polarographic data suggests that in the kinetics of metal cation/toxin complexation, a less stable intermediate complex was formed followed by a more stable secondary complex. The presence of multiple, potential binding sites made it difficult to estimate discrete formation constants but, nevertheless, log K, formation constants have been calculated which, at values of about 5, indicate that microcystins are medium-strength ligands. Substitution of arginine with either tryptophan or phenylalanine, i.e. replacement of MC-LR by MC-LW or MC-LF, appeared to have no discernible effect on the calculated formation

constants, suggesting that the side-chain groups of these amino acids either have a minor, shared role with other amino acids or do not participate in coordination with metals. Hence, the data demonstrated the presence and strength of binding but did not elucidate mechanisms or locate a single binding site, implying that a chelating effect occurs whereby the metal ion is bound by multiple sites on the peptide. If either copper or zinc were preferentially bound to a particular type of functional group present on the heptapeptide, then the overall complexation characteristics for each metal/microcystin system would be expected to differ. This was not observed and formation constants for both metals were similar. Calculation of formation constants is independent of metal concentration so that the two systems, copper/microcystin and zinc/microcystin, can be compared, even though the solubilities of the two metals differ. Published data for the complexation of amino acids with metal ions show that, in general, Cu 2÷ ions are more strongly complexed than Zn 2+ ions as reflected in K, values (Sillen and Martell, 1971). These two metals may not necessarily compete for the same sites. Conformational changes that occur when trace metals bind to the different sites may result in a competing or enhanced binding effect. Such an effect has been reported by Cao et al. (1995) in investigating the effects of competing trace metals and Ca 2+ on Cd 2+, Pb 2+ and Cu 2+ complexation by humic acids extracted from groundwater, determining the labile metal concentrations in these titrations by DPP. As a further example of enhanced binding, experiments with fungal polysaccharides have shown that Pb 2+ ions exhibit a higher affinity for oxidised scleroglucan than Cu 2+ ions as a direct result of disorder-to-order conformational transition of the polysaccharide backbone. Cu 2+ ions however were unable to induce this conformational transition and were less effective at binding (Bosco et al., 1995). Individual amino acids vary greatly in their complexation capacity with histidine, often found in these metal binding proteins and siderophores (Neilands, 1981) having the greatest K, value. The amino acids present in the microcystin heptapeptide, however, are all ligands of weak-to-intermediate strength (e.g. Fig. 1) and hence it might be expected that the overall complexation characteristics will reflect this. The microcystin concentrations used were the minimum necessary to produce a measurable effect and hence the shifts in evolution potential were small. The mathematical form of the F-functions is such that characteristic plots are produced which aid in establishing the number of complex ions formed in a given system and also provide a qualitative check on the validity of the data (Deford and Hume, 1951). Thus a plot of F,(X) against ligand concentration for the last complex will be a straight line parallel to the x-axis. A similar plot for the immediately preceding

Binding of copper and zinc to microcystins complex will be a straight line with a positive slope and all the previous F-function plots will show curvature. Such characteristic plots were observed in all cation/microcystila systems examined, confirming that the experimented conditions and the data were valid. At pH values above the isoelectric point the toxin will have a net r~gative charge, allowing ionic interaction with me~Lal cations. It can be predicted that complex formation in the polarograph system would be influenced by pH and this was observed in the case of copper/1VtC-LR complexes, which formed stable complexes at pH 7.5 but not at pH 5.5. This can be explained by greater protonation of the MC-LR molecule at lower pH values. In continuation of this work, proton displacement experiments similar to those performed by Crist el al. (1981) might confirm such a proposed coordination mechanism, since metal-binding and associated displacement of protons may result in a detectable decrease in pH. Complex formation between zinc and MC-LR did not appear to be influenced as strongly by the pH of the supporting electrolyte, as determined by the formation constants; calculated at each pH value. However, examination of plots of the normalised peak current against concentration revealed an unexpected inverse relationship between pH and reduction in zinc ion peak height, with the greatest decrease in peak amplitude being observed at pH 5.5. As low molecular weight peptides, microcystins are ideal molecules for analysis by polarography since larger macromolecules are inherently more likely to produce adsorption effects at the electrodes, interference and non-standard behaviour (Diaz-Cruz et al., 1992). Additionally, they are intermediate strength ligands which are most suited to this methodology and their cyclic stru,:ture may make analysis by other means difficult. By demonstrating metal-binding to microcystins, the work presented here has opened further avenues of investigation of these toxic peptides. The heal'Lh hazards presented by microcystins to mammal:; in acute and chronic exposures are beginning to be understood and their significance taken into account in waterbody management (Bell and Codd, 1994). 'The interactions between microcystins and metals with resulting complex formation, as presented in the present paper, merit toxicity assessment of the rnicrocystin-metal complexes. CONCLUSIONS Differential pulse: polarography was applied to the study of cyanobacterial microcystins. The strength of complexation between these cyclic peptides and copper and zinc was evaluated in terms of formation constants. The formation constants for microcystinLR, microcystin-LW and microcystin-LF at pH 7.5 indicate that they a:re intermediate strength iigands of copper and zinc and that substitution of amino acids at the arginine locus on the microcystin ring does not


significantly effect the strength of complexation. Complexation between microcystin-LR and copper appeared to be dependent on pH, with the extent of binding decreasing with decreasing pH of the supporting electrolyte. Acknowledgements--A. V. Humble thanks the Natural

Environment Research Council (UK) for a Ph.D studentship during these investigations and K. A. Beattie and C. White for their technical advice.


Ahner B. A. and Morel F. M. M. (1995) Phytochelatin production in marine algae. 2. Induction by various metals. Limnol. Oceanogr. 40, 658-665. Azevedo S. M. F. O., Evans W. R., Carmichael W. W. and Namikoshi M. (1994) First report of microcystins from a Brazilian isolate of the cyanobacterium Microcystis aeruginosa. J. Appl. Phycol. 6, 261-264. Beard S. J., Ciccognani D. T., Hughes M. N. and Poole R. K. (1992) Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media. FEMS Microbiol. Lett. 96, 207-212. Bell S. G. and Codd G. A. (1994) Cyanobacterial toxins and human health. Rev. Med. Microbiol. 5, 21-30. Birch L. and Bachofen R. (1990) Complexing agents from microorganisms. Experientia 46, 827-834. Bosco M., Sussich F., Gamini A., Reisenhofer E., Adami G. and Rizzo R. (1995) Divalent-cation interactions with a carboxylated derivative of scleroglucan. Macromolec. Chem. Phys. 196, 3979-3989. Cao Y., Conklin M. and Betteron E. (1995) Competitive complexation of trace metals with dissolved humic acid. Environ. Health Persp. 103, 29-32. Campbell D. L. (1994) Laboratory and field investigations into the cyanobacterial hepatotoxin, microcystin-LR. Ph.D. Thesis, University of Dundee. Carmichael W. W. (1994) The toxins of cyanobacteria. Sci. Am. 270, 78-86. Carmichael W. W., Beasley V., Bunner D. L., Eloff J. N., Falconer I., Gorham P., Harada K.-I., Krishnamurthy T., Min-Juan Y., Moore R. E., Rinehart K., Runnegar M., Skulberg O. M. and Watanabe M. (1988) Naming of cyclic heptapeptide toxins of cyanobacteria (blue-green algae). Toxicon 26, 971-973. Codd G. A., Brooks W. P., Lawton L. A. and Beattie K. A. (1989) Cyanobacterial toxins in European waters: occurrence, properties, problems and requirements. In Watershed '89. The Future for Water Quality in Europe.

Vol. 2 (Edited by Wheeler D., Richardson M. J. and Bridges J.) pp. 211-220. Pergamon Press, Oxford. Craig M., McCready T. L., Mark H. A. L., Smillie A., Dubord P. and Holmes C. F. B. (1993) Identification and characterisation of hydrophobic microcystins in Canadian freshwater cyanobacteria. Toxicon 31, 1541-1549. Crist R. H., Oberholser K. and Nguyen M. (1981) Nature of bonding between metallic ions and algal cell walls, J. Am. Chem. Soc. 15, 1212-1217. Deford D. D. and Hume D. H. (1951) The determination of consecutive formation constants of complex ions from polarographic data. J. Am. Chem. Soc. 73, 5321-5322. Diaz-Cruz J. M., Esteban M., van den Hoop M. A. G. T. and van Leeuwen H. P. (1992) Protolytic control in stripping voltammetric titrations of metal-polyacid complexes. Anal. Chim. Acta 264, 163-175. Filella M., Buftle J. and van Leeuwen H. P. (1990) Effect of physio-chemical heterogeneity of natural complexants. Anal. Chim. Acta. 232, 209-223. Gadd G. M. and Edwards S. W. (1986) Heavy metal-induced flavin production by Debaryomyces


A.V. Humble et al.

hansenii and possible connexions with iron metabolism. Trans. Br. Mycol. Sot'. 87, 533-542. Heath G. A. and Hefter G. (1977) The use of differential pulse polarography for the determination of stability constants. J. Electroanal. Chem. 84, 295-302. Horne A. J. and Goldman C. R. (1994) Limnology. Second edition. McGraw-Hill, New York. pp. 513-514, Katayama N., Nozaki Y., Okonogi K., Harada S. and Ono H. (1993) Ferrocins, new iron-containing antibiotics produced by bacteria. J. Antibiotics 46, 65-70. Kenefick S. L., Hrudey S. E., Peterson H. G. and Prepas E. E. (1993) Toxin release form Microcystis aeruginosa after chemical treatment. Wat. Sci. Technol. 27, 433-440. Laulhere J. P., Laboure A. M., van Wuytswinkel O., Gagnon J. and Briat J. F. (1992) Purification, characterisation and function of bacterioferritin from the bacterium Synechocystis PCC 6803. Biochem. J. 281, 785-793. Lawton L. A., Edwards C., Beattie K. A., Pleasance S., Dear G. J. and Codd G. A. (1994) Isolation and characterisation of microcystins from laboratory cultures and environmental samples of Microcystis aeruginosa and from an associated animal toxicosis. Natural Toxins 3, 50-57. Lingane J. J. (1941) Interpretation of the polarographic waves of complex metal ions. Chem. Rev. 29, 1-31. Lukac M. and Aegerter R. (1993) Influence of trace metals on growth and toxin production of Microcystis aeruginosa. Toxicon 31, 293-305.

Neilands J. B. (1981) Iron absorption and transport in microorganisms. Ann. Rev. Nutr, 1, 27-46. Ogiwara T. and Kodaira K. (1989) Measurement of copper complexing ability of the exudates of a Microcystis. War. Res. 23, 23-27. Riley T. and Watson A. (1987) Polarography and Other Voltammetric Methods. J. Wiley and Sons, London. Rinehart K. L., Namikoshi M. and Choi B. W. (1994) Structure and biosynthesis of toxins from blue-green algae (cyanobacteria). J. Appl. Phycol. 6, 159-176. Ruiz C., Mendieta J. and Rodriguez A. R. (1995) The electrochemical behaviour of Cd, Zn thioeins depending on the solution pH using differential pulse polarography. Anal. Chim. Acta 305, 285-294. Sillen L. and Martell A. E. (1971) Stability Constants. Special Publication no. 25, Royal Soc. Chem., London. Stanier R. Y., Kunisawa R., Mandel M. and Cohen-Bazire G. (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Revs. 32, 171-205. Utkilen H. and Gjolme N. (1995) Iron-stimulated toxin production in Microcystis aeruginosa. Appl. Environ. Microbiol. 61, 797-800. Watanabe M. F., Tsuji K., Watanabe Y,, Harada K. I. and Susuki M. (1992) Release of the heptapeptide toxin (mierocystin) during the decomposition process of Microcystis aeruginosa. Natural Toxins 1, 48-53.