Applications of enzyme-catalysed reactions in trace analysis—VI

Talanta, 1970, Vol. 17, pp. 299 to 304.

Pergamon Press. Printed in Northern Ireland

APPLICATIONS OF ENZYME-CATALYSED REACTIONS IN TRACE ANALYSIS-VI DETERMINATION OF MERCURY AND SILVER BY THEIR INHIBITION OF YEAST ALCOHOL DEHYDROGENASE* A. TOWNSHEND and A. VAUGHAN Chemistry Department, The University, P.O. Box 363, Birmingham 15, England (Received 6 October 1969. Accepted 9 November 1969) Summary-_The inhibition of yeast alcohol dehydrogenase is applied

to the determination of 2-200 ng of mercury(II) and l-100 ng of silver. The enzyme, when used to catalyse the oxidation of ethanol, or the reverse reaction, is equally sensitive to inhibitors. YEAST ALCOHOLDEHYDROGFINASE,one of many enzymes classed as alcohol dehydro-

genases,l is commonly available.

It catalyses the reaction

NAD+ + C,H,OH + NADH + CH,CHO + H+ where NAD+ is nicotinamide adenine dinucleotide. Optimal catalytic efficiency for the oxidation of ethanol2 occurs at pH 7-8, and that for the reverse reaction at pH 5.5-6.5. The enzyme can participate in the oxidation of a great many normal and branched-chain aliphatic alcohols and carbonyl compounds. It has a molecular weight of about 1.5 x 106 and each molecule contains 4 or 5 tightly-bound prosthetic zinc ions. The enzyme is inhibited by heavy metals,4-7 complexing agent&lo, certain coenzyme analogues, purines and pyridine derivatives.llJ2 The amount of metal required for inhibition follows the order Hg(I1) < Ag < Cd < Cu(I1) < Pb < Zn. Mercury and silver cause dissociation of the enzyme protein into sub-unitPJ4 with release of enzyme-bound zinc. Inhibition by complexing agents is a result of their complex formation with the prosthetic zinc ions. Among the species that inhibit in this way are 1,10-phenanthroline,1°~15~1a 2,2’-dipyridy1,1’ 8-aminoquinolinet8 8hydroxyquinoline,8 dithizone,8 thiourea and diethyldithiocarbamate.g This paper describes the investigation of the inhibition of yeast alcohol dehydrogenase by metal ions in order to develop sensitive means of analysis for these metal ions. It was particularly desirable to investigate the effect of these species on an enzyme catalysing a reversible reaction in the forward and reverse directions, in order to find what effect this might have on selectivity or sensitivity. Eflect of metal ions on the enzyme The catalytic activity of the enzyme on the reaction between 10-2M ethanol and 10-4M NAD+ was greatest at pH 8.0, whereas its activity in catalysing the reverse reaction, between 10dM NADH and 10T2ikf acetaldehyde was greatest at pH 6.5. The effects of added metal ions on the enzyme catalysis of the forward and reverse reactions are summarized in Table I and the variations of the effects with metal ion concentration are shown in Figs. 1 and 2. They con8rm that mercury and silver are * Part V, Tduntu, 1970, 17,289. 299

300

A. Tow~smND and A. VAUGHAN TABLEI.-EFFFCT OF METALIONSON YEASTALCOHOL DEHvDRoGENAsB Ethanol oxidation Metal *

No incubation Cont., M R.A.

HgO HgCI) Ag Cd Au(nI) ZD Cu FeW) Co(I1) Ni Bi CrW

2.7 4.2 1.0 7.6 5.0 1.0 1.2 1.3 1.3 1.3

x x x x x x x x x x -

10-O 1O-8 10-1 lo-* 10-1 10-b 10-S 10-d 10-d 10-d

1.00 050 0.50 050 050 0.50 0.50 0.50 060 0.42 0.04 -

Acetaldehyde reduction

Incubationt Cont., M R.A. 75 1.0 6.5 1.0 1.3 7.5 1.3 1.3 1.3 1.3 1.3

x x x x x x x x x x x

10-B 10-a lO-s 10-1

1.00 0.50 0.55 0.50 0.50

10-s IO-6 lo-’ lo-’ lo-’ 10-b 10-d

0;5 0.56 064 0.48 0.28 0.52 0.72

cont.,

M

5 x 10-a 1.3 x 10-0 1.3 x 10-e 1.3 -x 1.3 x 1.3 x 1.25 x -

. . pK6x 1.00 050 0% 0.12

10-o IO-6

G7 0.03

lo-’ lo-‘

Oq8 0.86 -

* Be, Pb, Ca, Sr, Ba, Mg, MD(H) (1.3 x 10-4it4) all gave R.A. = 1 GO. t In unbuffered water. $ Incubation. R.A. = Relative Activity.

very potent inhibitors, and that the inhibitory strength of the metal ions decreases in the order: H&II) > Ag > Hg(1) > Cd > Cu(I1) > Zn > Ni > Co(II), Fe(III), Bi, Cr(III), after incubation with the enzyme in unbuffered solution for 10 min. Incubation in unbuffered solution markedly increases silver inhibition, and also increases the effects of mercury and copper. It reduces the effectiveness of cadmium and nickel, but has little effect on any of the other metals studied. Incubation at pH 8.0 had little effect on metal ion inhibition. Furthermore, the same ions in the same concentration ranges had similar effects on the enzyme irrespective of whether it catalysed the forward or reverse reaction. I-Or

0.8 -

F

‘S

‘i

O-6-

8

E ._ t 0.4z 0.2 -

FIG. l.-Instantaneous

inhibition of alcohol dehydrogenase at pH 8.0 by (a) H&II); (6) Ag; (c) Cd; (d) AuO; (e) Cu(lI); 0 ZD.

301

Applicationsof ewyme-catalysedreactions--VI

PM FW

2.-Inhibition of alcohol dehydrogenaseby (a) H&II); (b) Ag; (c) Cd; (d) Zn; (e) Cu, after incubation in unbuffered solution.

The results indicated that the inhibition was likely to be useful in developing methods for the determination of mercury and silver at the lO-*M level, and cadmium The plots of relative activity VS. log metal ion and gold at larger concentrations. concentration (Fig. 1) show that simple calibration graphs for silver and mercury can readily be obtained. The graph for cadmium had a more complicated shape, so analytical applications were not pursued. Determination of silver and mercury

When the enzyme was incubated with silver or mercury(I1) at pH 8.0, reproducible inhibition was obtained, and methods were achieved for 20-200 ng of mercury(I1) and l&100 ng of silver in 8 ml of final solution. The results for mercury are given in Table II. TABLEIL-DETERMINATION

OF MERCURY(II) AFTER INCUBATION AT pH 8.0

Mercury taken, ng found, ng

20 24,20

60 100 140 62,72 96,120 140,132

180 196,190

Similarly, a more sensitive procedure involving incubation in unbuffered solution gave the results in Table III. When the reverse reaction was employed, i.e., reduction of acetaldehyde, at pH 5.6, similar concentrations [l-lo ng of silver, 2-20 ng of mercury(II) in 8 ml of final solution] could be determined. However, the reaction is more difficult to carry out under these conditions, and the results obtained are less reproducible than those obtained from the forward reaction. Attempts were made to mask mercury so that silver could be determined in the presence of mercury. The complexing agents investigated: EDTA (6.25 x lodM), 1,2-diaminopropanetetra-acetate (1.2 x 10-8M), ethylenediamine, diethylenetriamine, triethylenetetramine (1.3 x 10-8.&f) and dithizone (1.3 x lo-‘M) were unsuccessful in that they removed the prosthetic zinc from the enzyme, or complexed too strongly with silver for adequate discrimination to be made between the two

A. TOWN~HEND

302

and A.

VAUOHAN

TABLE III.--DETERMINATION OF MERCURY(II)AND SILVERAFAR INCUBATrONIN UNBUFFERED SOLUTION Silver taken, ng found, ng

0.06, :*:

Mercury taken, ng found, ng Mercury taken, ng found, ng

‘2.:, 2.4,

2.8

14.0 13.2,14-O, 14.2

3.3 3.3,3*7

5.5: l.5

4.0 4.6

6.0 8.9,9.8,6*4

8.0 7.8

16.0 16.0

18.0 18.0,lS.O

20.0 19.2

7.6 7.6,7.6

9.8 104,10*1 10.0 9.7, 9.8, 10.0

12.0 11.0

metals. Furthermore, the triamine and tetramine contained fluorescent impurities that interfered with the monitoring of the NAD+. DISCUSSION

Inhibition of enzyme systems by metals is a well-known phenomenon. Mercury and silver are particularly strong inhibitors of numerous enzymes, usually by binding to -SH groups, so it is not surprising that other enzyme systems have been applied to the determination of small quantities of these metals. It is valuable, therefore, to compare the sensitivity of the present system to mercury and silver with that attained in other enzymic inhibition procedures. This is done in Table IV. The present method is as sensitive for silver as the urease method, and ten times more sensitive for mercury. The other methods tabulated are less sensitive for both metals. TABLE IV.-SENSIFXIY

OF ENZYME-INHIBITION METHODS TO SILVER AND MERCURY

Range determined References

Enzyme Xanthine oxidase Urease Invertase Glucosidase Glucose oxidase Alcohol dehydrogenase

Silver

Mercury

1 l-50 pg l-54 ng 0.1-0.9 pg 20 ng-2 pg l-100 ng

20-120 pg 20-100 ng 40-170 ng 0.3-27 pg 04-l *6 ,ug 2-200 ng

22 23 24 25 26 *

* Present work.

The present investigation confirms the results of other worker9 regarding the effectiveness of various metal ions as inhibitors, the inhibition efficiency being Hg > Ag > Cd > Cu > Zn, both for the forward and reverse reactions. Lead, however, was found not to inhibit the enzyme, although lead has previously been reported as an inhibitor. s~17 Inhibition is thought to be due to binding of the metal ions with -SH groups on the enzyme,6’14 a view supported by the linear relationship between the solubility products of metal sulphides and the least concentration of these metals required to inhibit the enzyme completely. The activity of the enzyme increases with the number of free -SH groups. 14*lQInhibition by silver and mercury is also found to cause irreversible dissociation of the enzyme into four sub-units, possibly by removal of zinc from the enzyme. 13*14The reverse reaction was generally inhibited in the same way as the forward reaction, so that there would be little advantage in using it rather than the more reliable forward reaction. Nevertheless,

Applications

of vtalysed

reactions-VI

303

some differences in inhibitory effectiveness were observed (silver, for example, was appreciably less inhibitory towards the reverse reaction) which could be developed to provide a degree of selectivity. EXPERIMENTAL The enzymatic reaction was monitored by measuring the intensity of the fluorescence of the NADH formed or consumed in the reaction, in a lo-mm cuvette. The fluorimeter was calibrated with 10-6M quinine sulphate in @1M sulphuric acid to give a full-scale deflection at a sensitivity a tenth of that used to monitor the enzyme reaction.

Water distilled from an all-glass apparatus was used throughout. Enzyme. Yeast alcohol dehydrogenase (Seravac Laboratories, Maidenhead, Berks.) of nominal activity 220 pmole. mg-I. mml, was stored at 0” as a 0.006’~ solution in @lx bovine albumin sol~tion,*~ and was diluted 5-fold before use. The crystalline enzyme lost <5 % of its activity when stored at 0” for 4 months; the stock solution lost cu. 10 ‘A of its activity over 5 davs at this temperature. Tris(hydroxymethyl)methylatnine (tris) buffer. Prepared as de&bed previously,” except that it was adiusted to DH 8.0 with hvdrochloric acid. Act&e bufe;. Sodium a&ate was recrystallized in the same way as tris;*l 4.2 g were dissolved in water (400 ml), the pH was adjusted to 5.6 with MAR-grade hydrochloric acid, and the solution was diluted to 500 ml with water. Substrates. Aqueous 0.1 M solutions of ethanol and acetaldehyde and @01x solutions of NAD+ and NADH. Metal ion solutions. Stock 10-4M solutions were prepared from analytical-grade reagents. More dilute solutions were prepared immediately before use by appropriate dilution of these solutions. Adsorption of metal ions on glassware As mercury and silver are strongly adsorbed onto glass, this can lead to serious errors when ngamounts of these metals are being determined, both by removal of metal ions from solution, or by release into solution during a subsequent analysis using the same glassware. It is essential to avoid these sources of error. Adsorption is minimized by leaving the solution in contact with the glass for as short a time as possible. Contamination can be removed by soaking in 10-ao~ dithizone in carbon tetrachloride for at least 2 hr, followed by washing with acetone and finally with water. Determination of silver (l-10 ng) or mercury (2-20 ng) In a lo-ml flask mix accurately measured volumes of enzyme solution (1 ml) and silver or mercury(H) solution (2 ml). Place ih ice for 10 min. Add a soludon at 25” composed of solutions of NAD (3 ml), ethanol (1 ml) and tris (1 ml). After exactly 45 set (or other suitable time) measure the fluorescence intensity of the solution, and also that of a similar solution to which no enzyme has been added, and calculate the net increase in fluorescence intensity (Fi). Repeat the experiment in the absence of inhibitor, to obtain the net increase in fluorescence intensity due to the uninhibited reaction (F,), and calculate Ff/Fu, the relative activity. To construct a calibration graph, use solutions of known silver or mercury content, and plot F,/F,, vs. metal concentration. Determinatian of silver (10-100 ng) or mercury (20-200 ng) Use the same procedure, but add tris buffer solution (1 ml) before the incubation. Determination of silver (l-10 ng) or mercury (2-20 ng) by the reverse reaction In a lo-ml flask mix accurately measured volumes of enzyme solution (1 ml), acetate buffer solution (1 ml) and silver or mercury(I1) solution (1 ml). Place in ice for 10 min. Add 5 ml of a mixture at 3“ of solutions of NASH (1 ml), acetaldehyde (1 ml) and water (3 ml). Continue as in the procedures above, but measure the net decrease in fluorescence intensity. Again plot Fi/Fu VS. metal concentration to obtain a calibration curve. Experiments with other metal ions were carried out by the methods given above for silver and mercury. Acknowledgements-The authors thank Professor R. Belcher for his interest and encouragement: A.V. thanks the Chemistry Department for the provision of a maintainance grant.

A. TOWNSHENDand A. VAUGHAN Zusammenfassung-Die Inhibition von Alkohol-Dehydrogenase aus Hefe wird auf die Bestimmung von 2-200 ng Quecksilber(II) und l-100 ng Silber angewandt. Bei der Katalyse der Oxidation von Athanol oder der umgekehrten Reaktion ist das Enxym gegen die Inhibitoren gleich empfindlich. R&nnUn applique l’inhibition de la deshydrogenase alcoolique de levure a la determination de 2-200 ng de mercure (II) et de l-100 ng d’argent. L’enzyme, lorsqu’on I’utilise pour catalyser l’oxydation de l’&hanol, ou la reaction inverse, est egalement sensible aux inhibiteurs. REFERENCES 1. H. Sund and M. Theorell, in The Enzymes, P. D. Boyer, H. Lardy and K. Myrbiick, eds., Vol. 7,2nd Ed., Academic Press, New York, 1963. 2. A. P. Nygaard and H. Theorell, Actu Chem. Scar&, 1955,9,1300. 3. J. E. Hayes and S. F. Velick, J. Biol. Chem., 1954,207,225. 4. K. Wallenfels, H. Sund, A. Faessler and W. Burchard, Biochem. Z., 1957,329, 31. 5. K. Wallenfels, H. Sund, M. L. Zarnitx, 0. P. Malhoha and J. Fischer, in Sulphur in Proteins, R. Benesch and R. E. Benesch, eds., p. 215. Academic Press, New York, 1959. 6. R. A. Clayton, Arch. Biochem. Biophys., 1959, 85,559. 7. E. S. G. Barron, B. Miller, G. R. Bartlette, J. Meyer and T. P. Singer, Biochem. J., 1967,41,69. 8. B. L. Vallee and F. L. Hoch, Proc. Nat. Acad. Sci. U.S., 1955,41,327. 9. B. L. Vallee, Adv. Protein Chem., 1955,10,317. 10. F. L. Hoch and B. L. Vallee, J. Biol. Chem., 1956,221,491. 11. H. von Euler, Biochem. Z., 1936,286,72. 12. J. van Eys and N. 0. Kaplan, Biochim. Biophys. Acta., 1957,23,574. 13. H. Sund, Biochem. Z., 1960,333,205. 14. P. J. Snodgrass, B. L. Vallee and F. L. Hoch, J. Biol. Chem., 1960,235, 504. 15. F. L. Hoch, R. J. P. Williams and B. L. Vallee, ibid., 1958,232,453. 16. R. J. P. Williams, F. L. Hoch and B. L. Vallee, ibid., 1958, 232,465. 17. B. L. Vallee and F. L. Hoch, J. Am. Chem. Sot., 1955,77,821, 1393. 18. K. Wallenfels, H. Sund and H. Diekmann, Biochem. Z., 1957,329,48. 19. F. L. Hoch and B. L. Vallee, Arch. Biochem. Biophys., 1960,91,1. 20. E. Racker,J. Biol. Chem., 1950,184,313. 21. A. Townshend and A. Vaughan, Talanta, 1969,16,929. 22. G. G. Guilbault, D. N. Kramer and P. L. Cannon, Anal. Chem., 1964,36,606. 23. E. C. Toren and F. J. Burger, Microchim. Acta. 1968.1049. 24. D. Mealor and A. Townshend. Talanta. 1968,15,747, 1371. 25. G. G. Guilbault and D. N. Kr~amer, A&d. Bikhhm., i967,18,313. 26. E. C. Toren and F. J. Burger, Microchim. Acta, 1968, 538.