Applications of enzyme-catalysed reactions in trace analysis—IV

Talanta, 1969, Vol. 16, pp. 929to 937.

APPLICATIONS

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OF ENZYME-CATALYSED IN TRACE ANALYSIS-IV

REACTIONS

DETERMINATION OF BERYLLIUM AND ZINC BY THEIR INHIBITION OF CALF-INTESTINAL ALKALINE PHOSPHATASE A. TOWNSHEND and A. VAUGHAN Chemistry Department, The University, P.O. BOX363, Birmingham 15, England (Received 23 December 1968. Accepted 14 January 1969)

Summary-Methods are described for the determination of beryllium (18-90 ng) and zinc (06-6 ,ug), based on their inhibition of calfintestinal alkaline phosphatase. THE activation and inhibition of enzymes by inorganic species has recently received

some attention as a means of selective and sensitive inorganic trace analysis.lm4 This paper describes an investigation of the effects of metal ions and complexing agents on calf-intestinal alkaline phosphatase, and the subsequent development of selective analytical procedures for nanogram amounts of beryllium and microgram amounts of zinc. The enzyme There are many enzymes classified as alkaline phosphatases, which have markedly different properties. 5 Even those isolated from a single source, as in the present instance, are often mixtures of similar enzymes. The enzyme used in the present study is one of the commonly available intestinal mucosa enzymes, with optimal efficiency at pH 9-10. It catalyses the hydrolysis of phosphate esters (I) of many hydroxy compounds, including carbohydrates and phenols, and amines, also functions R-0-POa2-

+ Hz0 $ HOP0s2- + R-OH (I)

as a phosphotransferase between the substrate and, for example, tris buffer.6 The crystalline enzyme has been reported7 as having a molecular weight of 60000, and in common with alkaline phosphatases from other sources it contains zinc as an element essential for its activity.8 Zinc can be reversibly removed from alkaline phosphatases by this results in reaction with suitable ligands (l,lO-phenanthroline, EDTA, et+‘J); reduction or elimination of enzyme activity. The activity of alkaline phosphatases is usually enhanced by large concentrations ( 10V3M) of magnesium .ll Many metals have been reported to inhibit these enzymes, including beryllium,2*12J3 cadmium,12 cobalt(II),12 lead,12 copper(II),gJs zinc13J4 and bismuth,2 as have the pesticides aldrin and heptachlor.2 Indeed, when this investigation had almost been completed, Guilbault et aL2 reported the results of a similar, but less detailed investigation of hog-intestinal alkaline phosphatase. They developed methods for the determination of beryllium (0.01-30 ppm) and bismuth (l-70 ppm). * Part III, Talanta, 1968, 15, 1371. 929

A. TOWNSHENDand A. VAUGHAN

930

Their results, however, were significantly different in many respects from those described below. Where possible, comparisons between the results of the two investigations are made. EJ&ect of pH on reaction rate

The activity of the enzyme in catalysing the hydrolysis of ca. 10-3it4p-nitro-phenyl phosphate was greatest at pH 9.8, both in the presence of lO-4M magnesium, and in its absence. It was also found that the inhibitory effects of beryllium and zinc (see below) were greatest at this pH. In all instances, the extent of hydrolysis at pH 9.8 varied linearly with time for at least 10 min (the reaction time used in all subsequent experiments was 3 min). E#ect of added ions

The effect of added ions on enzyme activity with p-nitrophenyl phosphate as substrate is shown in Table I. The ions were not incubated with the enzyme before TABLE I.-EPPECTOFADDEDIONSONTHE R@LATIVEACTIVITYOFANALILUINE AT pH9.8FOR TWOSUBSTRATES

Mg2+

cw

Wf

NPP NP

140 1.26 1.23 Pb2+

140 1.39 1.03 Ni2+

140 140 1.07 1.16 1.00 1.00 Hg2+ VOz+

NPP NP

1 0.65 1.00 AiS+

140 0.61 0.84 BiSf

140 0.39 0.00 Bin+

;I;0

0.48 14

1.4 300 0.94 0.93 borate 300 0.83

Ion Cont., 10-SM RA for: Ion Cont., 10-6M RA for: Ion Cont., 10-5M RA for: Ion Cont., 10-6M RA for:

NPP oxalate NPP

300 0.40

30 0.97

Baa+

Mna+ Zn2+

Zna+

Ag+

14 1.24 1.10

1.4 0.31 0.79 Bea+

14 1.00 0.84 Ala+

voe+

14 0.12 0.11 Las+

140 0.00

14 0.38

14 o-97

I-

CN-

CN-

300 0.59

30 300 0.97 0.94 P043-

1000 0.14

300 0.13

100 0.38

1 0.97

30 0.86

0.14 0.50 0.75 CNS-

10 0.95

PHOsPHATAsE

14 0.82 0.72 Se-

EDTA 300 0.38

10 0.77

1 0.86

Cl-, Br-, IO,-, NOa-, NO*-, SOdz-, SzOae-, COQ2-, acetate (3 x 10-3M), F- (O*lM), Co”+ (1O-sM), Sn4+, Cr3+, Fes+ (10-4M) and Cu2+, Mna+, Fee+, Cd2+, VO*+ (10-KM) all gave RA = 1.00 with NPP. RA = relative activity; NPP = p-nitrophenyl phosphate, NP = 2-naphthyl phosphate.

substrate was added. It shows that beryllium and zinc are particularly potent inhibitors, although copper( vanadium(V), bismuth, lead, nickel, mercury(II), sulphide, cyanide and EDTA also have some inhibitory effect. Magnesium, the alkaline earth metals, and manganese(I1) all enhanced the activity of the enzyme. These results indicated that it should be possible to develop sensitive methods for beryllium and zinc on the basis of their inhibitory effects, although the ions named above would interfere and a suitable means of masking them would have to be found. Inhibition by beryllium

The change in relative activity of the enzyme in the presence of increasing concentrations of beryllium is shown in Fig. 1. It shows that the enzyme is more sensitive to beryllium when magnesium is present. However, the reproducibility of analyses for beryllium made under such conditions were poorer than those carried out in

931

Applications of enzyme-catalysed reactions in trace analysis--IV

Beryllium,

ng

I.--Inhibition of alkaline phosphatase by beryllium at pH 9-S; (a) in the absence of magnesium; (b) in the presence of 1.5 x 10-BM magnesium. Substrate, p-nitrophenyl phosphate. FIG.

the absence of magnesium. Thus the analytical methods were developed without adding magnesium to the enzyme. Under such conditions, 18-90 ng of beryllium in 7 ml of final solution could be determined accurately and reproducibly, in the absence of interfering species. Although small amounts of silver, cadmium and cobalt had no effect on the enzyme, it was found that 10”M concentrations of these elements slightly decreased the amount of inhibition caused by beryllium. There were many metals that also interfered by inhibiting the enzyme (copper, mercury, zinc, etc). It was necessary, therefore, to attempt to mask these species without removing the zinc bound by the enzyme, and causing inhibition anyway. An investigation of the effect of likely masking agents on the activity of the enzyme itself showed that diethyldithiocarbamate and fluoride had relatively little effect. The former ligand was applied in the beryllium determination; it completely masked the effects of at least 10”M silver, cobalt, nickel, copper and mercury, and 10-4M cadmium, in the final solution. In the procedure developed, therefore, the only metals that interfered were lead, vanadium, aluminium, manganese (cJ Table I), magnesium, and the alkaline earth metals at > 1.5 x 10m5M. TABLE II.-DETERMINATION OF BERYLLIUM

Be taken, ng Be found, II~ Be found, ngt Other cation present, 1.5 x lo-6M

45 47, 46, 4&

27 28.6, 29, 27

63 64, 63

81 78.r, 81

43

45

44

45

45

43

Ag+

Cda+

CdZ+*

NP+

co2+

Zna+

t 45 q of Be taken. * 1.5 x lo-4M. Inhibition by zinc

Although zinc is an essential component of alkaline phosphatases, when added in lug amounts it also inhibits the enzyme used here. The variation in relative enzyme activity 12

(RA) with changes in zinc concentration

is given in Fig. 2. Use of this graph,

A. TOWNSHEND and A. VAUCXUN

932

Fm. 2.-Inhibition

of alkaline phosphatase by zinc at pH P-8. phosphate.

or preferably, of the straight-line plot of log [(l - RA)/RA] ZIS.log [Znzt] as a calibration curve, permitted O-6-6 ~18of zinc in 7 ml of final solution to be determined accurately in the absence of interfering ions (Table III). Apart from expected interferences by other inhibitors, especially beryllium, by anions that complex with zinc and remove it from the enzyme (EDTA, sulphide, etc), and by metals that enhance the activity of the enzyme (alkaline earth metals erc), there was another source of interferen~. When the enzyme was i~bited by zinc, some other bivalent cations reactivated the enzyme. These metals included those that enhanced the activity of the unihibited enzyme (magnesium, alkaline earth metals, manganese) and lead. Aluminium, nickel and cadmium also had slight re-activating effects. In all instances, the relative activity of the enzyme was restored to the level that would have been attained in the absence of added zinc (e.g., to cu. 1.16 for barium). This reactivation has been applied to the determination of traces of barium.16 As lead was an inhibitor of the enzyme, it increased the enzyme activity only to that of the lead-inhibited enzyme. This is illustrated in Fig. 3. Fluoride was satisfactory for masking the effects of 1.5 x 1O-4M calcium, strontium and magnesium (Table III) and, if acetylacetone was also present, silver, nickel, TABLE III.-DETERMINATION OF ZINC

Zn taken, ,ug

Zn found, ,LJ~

065 0.65,@61

0.65 3.5 Zn taken, rug 0.75 3.5 Zn found, jg Other cation present, 1.5 x 10-4M Ca Ca

1.95 l-78, 1.95

3-2, 3.3, 3.2

5.8, 54,5*9&

4.:,sL5s

4.5, 4.8

1~9~ 1.8

32, 3.3

5.8 5.8

1*P5 l-5

3*28 3.2,

4.5, 4.6

Ca

Sr

Sr

Sr

Mg

Mg

Mg

Applications

of enzyme-catalysed

I -5.4

I - 5.0 log

933

reactions in trace analysis-IV

I -4.6

Pbl

FIG. 3.-Effect of lead on the relative activity of alkaline phosphatase at pH 9.8; (a) in the absence of added zinc; (b) in the presence of l-5 x 10-sMzinc. Substrate, p-nitrophenyl phosphate. and copper (l-5 x 10e4M) and beryllium (1.5 x 10-6M) did not interfere either. Lead, cadmium and barium (lObM) still interfered.

cobalt

2-Naphthyl phosphate as substrate

The results described above were obtained for the hydrolysis of p-nitrophenyl phosphate. The reaction was monitored spectrophotometrically by measuring the absorbance of p-nitrophenol formed during the reaction. Fluorimetric methods for monitoring enzyme reactions are usually more sensitive for enzyme assay,le so that 2-naphthyl phosphate, which hydrolyses to fluorescent 2-naphthol, was also investigated as a substrate. The effects of some cations on this system have been included in Table I. In general, the system was less sensitive to inhibition and activation. Indeed, strontium and barium did not activate the enzyme, and barium did not re-activate the zinc-inhibited enzyme. Only for mercury(II), silver and aluminium did the enzyme system become more sensitive. In most instances, therefore, 2naphthyl phosphate was an inferior substrate for the determination of trace metals. TABLE IV.-EFFECT OF 1.4 x 10-6M ZINC ON VARIOUSCONCENTRATIONS OF ENZYME*

Enzytie added, ,ug RA

22 0.80

* Substrate was 2-naphthyl

44 0.80 phosphate,

66 0.80

88 0.78

110 0.76

pH = 9.8.

It was also established that the relative activity of the enzyme partially inhibited by a given amount of zinc was independent of enzyme concentration when 2-naphthyl phosphate was used as the substrate (Table IV). This should be so if the inhibition can be represented by a simple equilibrium (E = enzyme) E + nZn2+ + EZn,.

934

A. TOWNSHEND

and A. VAUGHAN

The equilibrium constant k = [EZn,]/[E][Zn~]“; thus the relative activity, which is governed by the ratio [EZnJ [El, for a given zinc concentration, will be independent of the amount of enzyme added, provided that the latter is appreciably less than the amount of zinc present. Guilbault and his co-workers2 used umbelliferone phosphate at pH 8.0 as a ffuorogenic substrate in a similar investigation. Again, there are significant differences in the response of the system to metal ions, although there is insufficient information given to make a detailed comparison. Guilbault’s system appears to be less sensitive to beryllium but more sensitive to bismuth than the present procedure using p nitrophenyl phosphate. It is also less sensitive to aluminium, lead, copper and mercury. Zinc, up to at least 1O-4M, has no effect on the enzyme. It has previously been shown17 that the effect of zinc depends on the substrate, and upon enzyme purity, incubation time and other parameters. Also, alkaline phosphatases from some other sources e.g., E. coli are not inhibited by zinc.12 DISCUSSION

The mechanisms of inhibition and activation of alkaline phosphatases have been discussed by many authors. 18-20 It is suggested that the hydrolysis reaction is catalysed by the enzyme in a two-step reaction21: E-OH + R-O-P0,2+ E-0-P0s2+ R-OH E-0-POa2+ H,O + E-OH + HP042-. The relative rates of the reactions depend on the pH, but at fairly low pH values the phosphorylated enzyme can be isolated. Degradation of the enzyme then produces an 0-phosphoryl serine, suggesting that serine is involved in the enzyme active site.22.23 Photo-oxidation studies suggest that the zinc is bound by three histidine residues.lg Thus the active site could include the serine hydroxyl group and a zinc with one free co-ordination position:

YME Hydrolysis might occur via an intermediate in which the substrate is bound to the serine and the zinc ion. The effects of metal ions are so obviously dependent on the substrate that any mechanism devised to explain their effects must take this into account. It is possible that zinc (or other metal) that inhibits the enzyme is bound by the serine hydroxyl group and perhaps another co-ordinating centre in the protein. This prevents the phosphorylation of the enzyme. Examples in which inhibition does not occur can then be explained by the capacity of the substrate to compete successfully with the inhibiting metal for the serine hydroxyl group. Inhibition by other metals does not

Applications of enzyme-catalysed reactions in trace analysis-IV

935

involve replacement of the enzyme-bound zinc by the other metal,12 because zinc itself is an inhibitor. Activating metals such as magnesium seem to enhance enzyme activity by catalysing the first step in the enzyme reaction sequence. This is thought to be due to the formation of a substrate-magnesium complex. * Similarly, re-activation indicates that the substrate-re-activator complex is now strong enough to compete with the inhibiting metal for the serine hydroxyl group. Thus, if a metal does not activate the hydrolysis of a particular substrate, i.e., it does not complex with the substrate, it is unlikely that it will re-activate the inhibited enzyme, as has been shown above. Lead is an exception because although it is a re-activator, it inhibits the enzyme. It is possible, nevertheless, that a lead-substrate complex could function similarly to a magnesium-substrate complex. However, it is difficult to explain why further addition of lead then causes reversion to inhibition. Inhibition by beryllium has never been satisfactorily explained. In the present investigation, although the system is very sensitive to beryllium, the maximal inhibition attained, both in the presence and absence of magnesium, is 50 %. The concentration of beryllium required to achieve this is only 6-10 times that of the enzyme. The form of the inhibition plot (Fig. 1) tends to confirm strong enzyme (or substrate) binding with beryllium. Thomas and Aldridge20 have investigated the mechanism of beryllium inhibition, and have pointed out that many enzymes that are not inhibited by beryllium are also phosphorylated on serine, so that the simple inhibition model proposed for other metals is inappropriate. We feel that the inhibition and activation of alkaline phosphatases is such a broad and complex subject, and one that has spawned such a vast collection of unrelated and, often, insufficiently detailed observations, that any further speculation in the absence of unequivocal experimental evidence is almost useless. A long, careful, detailed study will be necessary before any reliable mechanisms for these processes can be formulated. The independence on enzyme concentration of the relative activity of an enzyme inhibited by a given amount of inhibitor is important for the analysis of inhibitors and activators. It shows that there will often be little or no advantage in using the more sensitive methods of enzyme assay because there will be no increase in the sensitivity of the system to inhibitors and activators. The experiments have shown that the effects of metal ions on an enzyme containing a readily-removable metal ion (zinc) as an essential metabolite can be used for the analysis of trace amounts of these metals. However, the problems of masking potential interferents are greater than with metal-free enzymes because the essential metal must not be removed from the enzyme by the masking agent. This has greatly restricted the range and concentration of possible complexing agents that can be used in this way. Nevertheless, sensitive, selective and reproducible methods for the determination of beryllium, barium and zinc have been devised. EXPERIMENTAL The hydrolysis ofp-nitrophenyl phosphate was monitored by measuring at 410 nm the absorbance of thep-nitrophenol formed in the reaction. The hydrolysis of 2-naphthyl phosphate was monitored by measuring the fluorescence of the 2-naphthol formed in the reaction, in a IO-mm cuvette, relative to 10-6M quinine sulphate in 0.1M sulphuric acid.

936

A. TOWNSH~NDand A.

VAUCXAN

Reagents Water distilled from a glass apparatus was used throughout. Enzyme. Calf-intestinal alkaline phosphatase [Serevac Labs. (Pty) Ltd., Maidenhead, Berks U.K.], of nominal activity 0*54pmole/mg/min, stored at 0”, used as 0.011% aqueous solution. The crystalline enzyme lost <5 % of its activity over 5 months at 0”. A 0011% solution of newly acquired enzyme lost <3 % of its activity over 5 days at 25”; some proteins then precipitated. However, as the crystalline enzyme aged, the protein precipitation occurred earlier; it required only a day after 5 months. Tris(hydroxymethyl)methylamine (tris). Recrystallized from an aqueous solution containing 10 mg of disodium-EDTA in 200 ml, and then from water. Buffer solution, pH 98, prepared by dissolving 605 g of tris in 400 ml of water, adjusting to pH 98 with MAR-grade hydrochloric acid and diluting to 500 ml with water. Substrates. Aqueous solutions (5 x lo-‘M) of their disodium salts. Sodium diethyldithiocarbamate. A 10maM aqueous solution. Acetylacetone-fluoride. Acetylacetone, 0.02% v/v in O*lM sodium fluoride. Metal ion solutions. A stock 10-*&f beryllium solution was prepared from analytical-reagent grade beryllium sulphate. Other beryllium solutions were prepared daily by appropriate dilution Other metal ion solutions were prepared from analytical-grade reagents as required. Procedures Determination of beryllium. In a lo-ml flask mix accurately measured volumes of p-nitrophenyl phosphate solution (3 ml), tris buffer solution (1 ml), beryllium solution (1 ml, containing 18-90 ng of bervllium) and sodium diethvldithiocarbamate solution (1 ml). Place in a thermostat at 25’ for 10 n&, and add enzyme solution at 25O(1.00 ml). Quench the reaction exactly 3 min after adding the enzyme, by adding 0*5M sodium hydroxide (1.00 ml), and measure the absorbance of the solution in a lo-mm cuvette at 410 nm against a blank that has been taken through the procedure but to which no enzyme has been added. From the results obtained for suitable standards plot a calibration curve of RA us. beryllium concentration (RA = net absorbance from inhibited reaction + net absorbance from uninhibited reaction). Note. The blank accommodates the changes in absorbance due to the formation of coloured or colloidal diethyldithiocarbamate complexes. Determination of zinc. Use the procedure for beryllium, but add zinc solution (1 ml, containing 06-6 pg of zinc) instead of beryllium solution, and use the acetylacetone-fluoride solution in place of the diethyldithiocarbamate solution. Plot a calibration curve of log [(l - RA)/RA] us. log [Zn] from the results obtained for suitable standards. Investigation of the effects of other ions was carried out by these procedures. The procedure used for 2-naphthyl phosphate was the same as described above, except that masking agents were not used. Acknowledgements-The authors thank Professor R. Belcher for his interest and encouragement; A. V. also thanks the Chemistry Department for the provision of a maintenance grant. The authors also thank Professor G. G. Guilbault for providing a copy of his paper some time before it was published. Zusannnenfassnn8-Verfahren zur Bestimmung von Beryllium (18-90 ng) und Zink (06-6 pg) werden beschrieben, die auf der Inhibition der alkalischen Phosphatase aus Killberdarm beruhen. R&urn~On d&it des methodes pour le dosage du beryllium (1890 ng) et du zinc (06-6 ,@ ba&es sur leur inhibition de la phosphatase alcaline de l’intestin de veau. REFERENCES 1. 2. 3. 4. 5.

G. G. Guilbault, Anal. Chem., l966,38,527R; l968,40,459R. G. G. Guilbault, M. H. Sadar and M. Zimmer, Anal. Chim. Acta, 1969, 44, 361. D. Mealor and A. Townshend, Talanta, 1968, 15,747, 1371, 1477. E. C. Toren and F. J. Burger, Mikrochim. Acta, 1968, 538, 1049. E.g., T. C. Stadtman in The Enzymes, P. D. Boyer, H. Lardy and K. Myrback, Eds., Vol. 5, p. 55. Academic Press, New York, 1961. 6. I. B. Wilson, J. Dayan and K. Cyr, J. Biol. Chem., 1964,239,4182. 7. G. Schramm and D. Armbruster, Z. Naturforsch., 1954,9b, 114.

Applications 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

of enzyme-cataiysed

reactions in trace analysis--IV

937

L. Engstrom, Bioehem. Biophys. Acta, 1961,52,36. J. C. Mathies, J. Biol. Chem., 1958,233, 1121. D. J. Plocke, C. Levinthal and B. L. Vallee, Biochem., 1962,1, 373. R. K. Morton, Biochem. J., 1957,65,674. D. J. Plocke and B. L. Vallee, Biochem., 1962,1,1039. R. S. Grier, M. B. Hood and M. B. Hoagland, J. Biof. Chem., 1949,180,289; F. W. Kfemperer, J. M. Miller and C. J, Hill, J. Biol. Chem., 1949, 180,281. R. K. Morton, Methods in ~zymulugy, S. P. Colowick and N. 0. Kaplan, Eds., Vol. II, p. 533. Academic Press, New York, 1955. A. Townshend and A. Vaughan, Anal. Letters, 1968,1,913, G. G. Guilbauit in Fluorescence Theory, instrumentation and Practice, G. G. Guilbault, Ed., p. 297 ff. Arnold, London, 1967. E. Hove, C. A. Elvehjem and E. B. Hart, J. Biol. Chem., 1940, 134,425. J. H. Schwartz, Proc. Nut. Acad. Sci. U.S., 1963,49,871. T. C. Bruice and S. Benkovic, Bioorganic Mechunisms, p. 67 ff. Benjamin, New York, 1966. M. Thomas and W. N. AIdridge, Biochem. J., 1966,98,94. R. K. Morton. Disc. Far&v Soe.. 1955.20.149. J. H. Schwartz and F. Lipm&m, Pioc. &at. .&ad. Sci. U.S., 1961,47,1PP6. L. Engstrom, Biochim. Biophys. Acta, 1962,56,606; Arkiv Kemi, 1962,X9,129.