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Applications of enzyme-catalysed reactions in trace analysis—VII
Talortrt~ Vol
21. pp 401-409
Perpamon Press. 1974 Prmted m Great Brttam
APPLICATIONS OF ENZYME-CATALYSED REACTIONS IN TRACE ANALYSIS-VII* DETERMINATION OF LEAD AND INDIUM BY THEIR INHIBITION OF ISOCITRATE DEHYDROGENASE R. A. SHEIKHand ALAN TOWNSHEND Chemistry Department, The University. P.O. Box 363. Birmingham 15. England (Received
10 Srprernher 1973. Accepred
21 November
1973)
Summary-The Inhibition of isocitrate dehydrogenase by lead. indium and calcmm has been studied. Methods for the determination of 0.024-l pegof lead and indium. based on this inhibition. are outlined. Lead may be separated from some interfering ions by extraction with tributyl phosphate in isobutyl methyl ketone. before application of the enzymic reaction.
Isocitrate dehydrogenase [isocitrate : NADP oxidoreductase (decarboxylating), EC. 1.1.1.42], ICDH, catalyses the oxjdative decarboxylation of isocitrate to a-ketoglutarate. A nicotinamide nucleotide is the co-enzyme used as the oxidant and a doubly-charged cation such as Mg2+ or Mn’+ 1srequired to activate the enzyme: CH,COOH
CH,COOH
I
I
HC-COOH
+ NADP+
Mn”,ICDH
HCH
\
I
I
+
NADPH
+ H+ + CO2
C-COOH
HC-COOH I
/
OH
0 (a-ketoglutaric
(isocitric acid)
acid)
The reaction proceeds from isocitrate to x-ketoglutarate through an enzyme-bound oxalosuccinate intermediate.’ There are two isocitrate dehydrogenases.2 One requires NADP as its co-enzyme whereas the other requires NAD. The enzyme used in the present study is the purified NADP-linked enzyme, obtained from pig’s heart.3-” molecular weight 61 x 103. It is inhibited by a wide range of metal ions.(’ and is activated by manganese(H). magnesium, cobalt(II) and zinc ions.(’ The enzyme has been used to determine klg amounts of magnesium in blood serum? and ng amounts of manganese, ’ by means of their activation property. An interesting method of titrating a sequence ofmetal ions (including Mg’+. Mn’+, Zn’+. Co’+, Cu’+, Cd’+ and Ni2’) with EDTA. using the activation and inhibition effects to follow the progress of the titration. has also been developed, using this enzyme.6 Earlier workers showed that ICDH was inhibited by ng amounts of a number of metal ions. This paper reports an investigation of the inhibitory effects of lead, indium and calcium on ICDH. with a view to developing sensitive methods for the determination of these * Part VI’ Thrm
1970. 17, 299 401
402
R. A.
SHEIKHand ALAN TOWNSHEND
elements. As many other ions would interfere in such determinations, either by inhibiting or activating the enzyme, attempts are made to utilize chemical separation techniques to improve selectivity. In the discussion that follows, all metal ion concentrations refer to the final solution in which the enzyme-catalysed reaction takes place, unless otherwise indicated. o,so 4
f 0
;
.cp
0.45
8 e, $ 8
0.40
I
I
I
I
5
IO
I5
20
Incubation
time,
mm
Fig. 1. Effect of incubation time of ICDH with (A) 20 ng of PbzC per ml in pH 83 tris buffer and (B) with 20 ng of In3 * per ml in unbuffered water.
INVESTIGATION
Efict
OF
EXPERIMENTAL
CONDITIONS
of manganese(IZ) ions and pH on the activity of ICDH
NADP-linked isocitrate dehydrogenase is active only in the presence of an activating metal ion. It is most effectively activated by manganese(I1) ion.8 and these were used in the present investigation. It was necessary to establish the manganese(I1) concentration and contact time with the enzyme required to induce maximal enzyme activity, under the conditions used for the metal ion determinations. On incubation of the enzyme with 6 x lo- 5 M manganese(I1) at 0” in 0.05M Tris buffer (pH 7.5) or water, the activity increases slightly at first but there is no further increase in activity after 10 min. As maximal inhibition of the enzyme by lead or indium is achieved after 15 min incubation of inhibitor with ICDH (Fig. l), this incubation time was used for both activator and inhibitor in all subsequent experiments. The effect of manganese(I1) concentration on enzyme activity, after incubation in pH 75 Tris buffer solution for 15 min, is shown in Fig. 2. Under these conditions, which are those recommended for the determination of indium (see below), greatest enzyme activity is stimulated by lo- ‘M manganese(I1). The same concentration of manganese(I1) was found to be necessary to give maximal activation under the conditions recommended for the determination of lead (pH 8.5 Tris buffer, cu. 0.13 units of enzyme per ml of reaction mixture). The change in enzyme activity with pH (Fig. 3) shows that maximal activity occurs at pH 7.4.
Enzyme-catalysed
o.44
403
4.5
$0
5.5
Fig. 2. ElTect of Mn2+ concentration
reactions
on ICDH activity after 15 min incubation in pH 7.5 Tris buffer.
Inhibition by lead As indicated above, inhibition by lead is maximal after 15 min at O”, and is extensive even after 1 min. Incubation is best done at o”, because the enzyme slowly loses activity at 25”. Figure 3 shows that inhibition is greatest at pH 8.5. Thus, combination of these conditions gives the greatest sensitivity to lead reproducible inhibition being obtained over the range 2-70 ng of lead per ml (Fig. 4). Kratochvil et aL6 have reported that NADP-linked ICDH is inhibited by traces of many metal ions. The effect on enzyme activity of incubating the enzyme with lead (20 ng/ml) and other ions (2 pg/ml) in pH 8.5 OOSM Tris buffer was studied. The results are summarized in Table 1. All the ions investigated interfered, by inhibition (high results) or activation I
.o-
0.75J s 0 5 a
0.51
I
I
I
7.0
e.0
I
I 9.0
PH
Fig. 3. Effect of pH on (a) ICDH activity; (b) inhibition by 40 ng of In”+ per ml; (c) inhibition by 20 ng of Pb2+ per ml.
404
R. A.
SHEIKH
and
ALAN TOWNSHEND
Calcium concentration,
Indium
fig
or lead concentration,
/ml
q/ml
Fig. 4. Inhibition of ICDH by various concentrations of (a) Pb”. (h) Cal’. under the recommended conditions, and of It?+ after incubation in (c) unbuffered water: (d) pH 75 Tris buffer solution.
(low results) except for Al 3+ . Complete inhibition of the enzyme was observed in the presence of Hg ‘+, Ag+, Zn2+, Cu2+ and Cd 2+ . Most of these results are in agreement with the effects noted previously.6 In an attempt to develop a selective method for lead, the extraction method of Yadav and Khopkarg was investigated. Tributyl phosphate (TBP, 30% solution in isobutyl methyl ketone) is reported to separate lead selectively from a wide range of metal ions’ in a medium that is 3M in hydrochloric acid and 2M in lithium chloride. The most probable composition of the extractable lead species is PbC12*2TBP.’ Lead can readily be stripped from the organic phase with water, for enzymic determination. The final aqueous extracts were flushed with a slow stream of nitrogen to get rid of the last traces of isobutyl methyl ketone, which was found to inhibit the enzyme. With use of this method lead (45 /lg) could be determined in the presence of up to 4.5 mg of Bi3+, &I+. and Cal+. 2.25 mg of Hg2+. and 1.1 mg of Ag+. These are the maximum tolerance limits as reported by Yadav and Khopkar,9 and greater ratios were not investigated. The results are shown in Table 2. Itlhihition by in&m Conditions for maximal inhibition of ICDH by indium were found in a similar manner to those for lead. With incubation in unbuffered water for at least 15 min. and the enzyme reaction carried out at pH 7.5. fairly reproducible inhibition was achieved over the range Table I. Direct determination of lead (20 q/ml) m the presence of other ions (2 jcgiml)
Enzyme-catalysed Table 2. Determmation
Other cations. (pg.irnl) Pb’+ found, ng/rnl
reactions
405
of lead after extraction with 30% tributyl phosphate in isobutyl methyl ketone (Pb*’ taken = 15 n&ml) 15
Bi3’ (1.6) 13 16
Ba2+ (1.6) 16 13
Ca’+ (1.6) 13 16
Hg’+ (0.8) 13 16
Ag’ (0.4) 16 13
2-l 00 ng of indium per ml (Fig. 4). Results for the determination of indium in pure solution are shown in Table 3. When the enzyme was incubated with indium in pH 7.5 buffer solution for 15 min, the inhibition was slightly less sensitive (Fig. 4) allowing 3-100 ng indium per ml to be determined. Determinations were more reproducible, however (Table 3). In order to eliminate the interference of other metal ions, an attempt was made to determine indium after solvent extraction separation. The procedure of Khosla and Rae” using extraction by N-benzylaniline in chloroform from a hydriodic acid medium was investigated. The extraction procedure is simple and the separation of the organic and aqueous phases is very rapid. Indium is later stripped from the organic phase with 2M hydrochloric acid. When a 9”/, w/v solution of N-benzylaniline in chloroform, reported by Khosla and Rae” to separate indium from most metal ions, was used, some benzylaniline appeared in the final aqueous solution, resulting in the complete inactivation of the enzyme. To overcome this problem the effect of smaller benzylaniline concentrations was investigated. It was found that the concentration had to be decreased to 1% to prevent inhibition by the extractant. However, when a lOO-fold amount (by weight) of some other ions was present with 60 pg of indium. and the 1% extractant solution was used, high results for indium were obtained (Table 4). Complete inhibition was observed after extraction when mercury and cadmium ions were present in lOO-fold amount.
The incubation of the enzyme with calcium was done in unbuffered water for 15 min. The pH of the reaction solution was the same as for the determination of indium. The method allowed the determination of 0.5-10 pg calcium per ml in the absence of interfering ions (Fig. 4). Stahilitj* of‘ the enl!rtle-ilzhihitor complexes
The reaction between a cationic inhibitor (M”+) and an active site of an enzyme, (E), can be represented by the equation E + rrMX+= M,“+. E
(1)
where II is the number of metal ions bound per active site.” Table 3. Determination No huff27
O.OSM
7-m
bufl?r. pH 7 5
of indium
Taken, rlg/rvl 2 Found. ng/rnl 3
5 7
10 13
30 36
80 85
Taken. ng/rn/ Found. ng/rnl
10 11
20 23
40 40
60 61
80 77
100 100
R.
406
Table 4. Determination Other cations In3 + found ng/mf
of
A.
SHEIKH and ALAN TOWNSHENII
indium after extraction with 194 N-benzylaniline ng/ml; other ions = 2 fig/ml) _ 19
20
Pbl+ 47
ca” 89
Co”+ 47
Al3f 39
in chloroform. (Indium taken = 20
&2+ 89
Ni” 58
Mg2’ 28
&‘f 39
Cd”+ H$+ >200 3200
An equilibrium constant, R, is given by K = CUW’I” CM;+ . E]
(2)
The relative activity, RA, is proportional to the concentration of free enzyme sites, [El. Therefore. the ~on~ntration of inhibited enzyme, CM;’ . EJ, is proportionai to (I- RA) so 1-RA _ CM:+ .E] RA CEj
(3)
where RA is the fractional enzyme activity relative to that of the uninhibited enzyme under the same conditions. A combination of equations (2) and (3) gives
1-RA _ CM”‘]” RA
l-RA
log RA
K
= n log CM”‘] - log K
t51
A plot of log (1 -RA)/RA us. log CM”‘] should therefore give a straight line of slope n and intercept log K. The results of such plots for lead, indium and calcium are summarized in Table 5. All three plots give straight lines and the values of n are all approximately unity, indicting that one ion per active site is responsible for the inhibition. The equilibrium constants confirm that the strength of binding by the enzyme increases in the order Ca2+ Q In3+ < Pb2+. Table 5. Equilibrium constants for the enzyme-inhibitor
Inhibitor In3 +
Incubation, PH 7.5 HtO*
I1
complexes
K,ll?Ok/l.
I.15 I.20 091 0.96 @90
64 6.0 3.4 4.3 24
x x x X x
lo-’ lo-’ to-’ lo-’ lo-’
Pb’ +
8.5
1.04 l-16 1.20 0.94 1.04
1.1 X I.3 x I.3 x 1.5 X 1.4 x
ca2+
H1O*
I .07
I.2 X 10-d
* II nhullrcd.
to-7 lo-’ 1o-7 lo-’ IO-’
Enzyme-catalysed reactions
407
Adsorption of metal ions on glassware
Traces of metals are readily adsorbed by glass surfaces. 12*13*I4 During these investigations. it was confirmed that lead is adsorbed by glass, which could lead to serious errors when ng amounts of this metal ion were being measured. This source of error could be
eliminated, however, by putting the enzyme solution into the incubation flask first, followed by the lead solution. The lead was preferentially bound by the enzyme, and no significant amount was lost on the glass surface. To minimize any possible contamination from adsorbed lead or other metal ions, however, all glassware was soaked before use for several hours in a saturated aqueous solution of disodium EDTA, and rinsed thoroughly with water. Adsorption was also minimized by leaving metal ion solutions in contact with the glass for as short a time as possible. As polyethylene is not so susceptible to these adsorption effects,” all stock metal ion solutions were stored in polyethylene bottles.
The methods described allow the reproducible determination of ng amounts of indium and lead and rather larger amounts of calcium. As the enzyme studied is also inhibited by a wide range of other metal ions, means of prior separation of indium and lead from potential interferences were sought. Solvent extraction was successfully applied for the separation of lead, provided no significant amount of extractant found its way into the presence of the enzyme. Isobutyl methyl ketone and N-benzylaniline both inhibited ICDH, but whereas the ketone could readily be eliminated from the final aqueous solution, and thus did not interfere in the determination of lead, N-benzylaniline could not be prevented from finding its way into the final aqueous solution, if its concentration was to be sufficient to separate indium from other metals. EXPERIMENTAL The enzymic reactions were momtored m IO mm silica cuvettes in a Beckman DB spectrophotometer suring at 340 nm the rate of formation of NADPH at constant temperature (25.0 k @lo).
by mea-
Rragmts
Water distilled from an all-glass apparatus was used throughout. En-_~~ne.Highly purified pig’s heart isocitrate dehydrogenase in 50% glycerol (Sigma Chemical Co.) of activity 5 fitmole. mg- ‘. min- ’ was stored at 4”. A standard solution was prepared from this stock enzyme daily before use by diluting. with 0.05M Tris buffer of the required pH, 20-fold v/v for indium and calcium determinations (pH 7 5) and lo-fold for lead determinations (pH 8.5). The standard enzyme solutions were kept in an ice-bath during use. T~rs(h~dro.~~r~~~t/~~~)~~~~t/~~~at~~~t~~ (Tris) bufir solution, DOSM. Tris (3.03 g) was dissolved in about 400 ml of water. adjusted to the required pH with 0.1&f analytical-grade hydrochloric acid and diluted to exactly 500 ml wnh water The solution was kept m a thermostat at 25” before use. Substrates. A reaction mixture consisting of 30 mg of analytical-grade,trisodium or_-isocitrate dihydrate (ICA) and I7 mg of the monosodium salt of nicotinamide adenine dinucleotide phosphate (Sigma Chemical Co.) in 25 ml of 0.05M Tris buffer solution of the required pH was used. This mixture was kept in an ice-bath during use and kept frozen when not in use. When frozen it was stable for weeks. Mrtal ion solutions. Stock solutions of indium and lead (200 pg/ml), calcium (120 &ml) manganese (5.0 x 10-‘M) and possible interfering ions (ca. 1 mg/ml) were prepared from analytical-grade reagents and stored m polyethylene bottles. More dilute solutions were prepared immediately before use by appropriate dilution of the stock solutions. Procedures D~~tcwmotrort ofleud (24-840 y). To a 2 ml volumetric flask add Tris buffer (pH 8.5. ca. 1.0 ml) and exactly 0.1 ml (0.1 mg of protem = 0.5 units) of enzyme solution previously cooled m ice. Mix gently and add
408
R. A.
S&H
and
ALAN TOWNSHEXD
I.2 x IO-‘M manganese solution (exactly 0.1 ml). Mix the contents of the flask aeain and add ~0.5 ml of lead solution, at O”,containing 24-840 ng of lead. Make up to the mark with pH 8.5 T& buffer at 0-. trux thoroughly and place in ice for 15 min. Add exactly 0.5 ml of this solution to a mixture of ICA-NADP in pH 85 Tris buffer (05 ml), pH 8.5 Tris buffer (1.0 ml) and water (1-Oml) all at 25.0”. After exactly 15 min at this temperature. measure the absorbance (Ai) at 340 nm in a 10 mm cuvette against water. Repeat the experiment in the absence of the inhibitor to obtain the absorbance (A,) resulting from the uninhibited reaction and calculate the percentage inhibition (%I = IOO(A,,,-A#&). Plot a calibration curve of ‘41 us. lead concentration for the results obtamed from standard lead solutions taken through the procedure. A new calibration curve should be made daily. Liquid-liquid extraction of lead with triburylphosphare. This was investigated as follows. To 1 ml of solution containing 45 pg of lead were added (4 ml of solution containing < 4.8 mg of Bi”‘. Ba” or Ca”. 7.4 mg of Hg’+ or I.2 mg of Ag+). To this solution, in a 100 ml separating-funnel. I2M hydrochloric acid (5 ml) and 4M lithium chloride (10 ml) were added, to make their respective concentrations 3M and 2:M In a total volume of 20 ml. The mixture was extracted with 10 ml of 30’4 tributyl phosphate in isobutyl methyl ketone b! shaking briskly for 5 min. The layers were allowed to separate, the aqueous layer carefully removed. and lead stripped from the organic phase by shaking with water (two 10 ml portions). The aqueous lead solutions were quantitatively transferred to a 25 ml volumetric flask and made up to volume with water. Isobutyl methyl ketone was removed from the aqueous solution by passage of a slow stream of nitrogen for 15 min. Aliquots of this aqueous solution (0.1 ml contained 0.18 pg of lead) were used for the enzymic determination of lead. Determinarion ofindium (24-1200 ng). Use the procedure described for lead, but incubate the enzyme (0.1 ml containing 50 pg of protein = 0.25 units) in water instead of tris buffer solution and add < 0.5 ml of solution containing 24-1200 ng of indium. Carry out the enzyme-catalysed reaction in pH 7.5 Trls bufier solution at 25.0 f 0 1”.Plot a calibration graph of%1 us. indiumconcentration. A new calibration curve should be made daily. Separation of indium by solvent extracrion with N-benzyluniline. The investigation was made as follows. In a 50 ml separatory funnel distilled water (6 ml). indium solution (I ml containing 60 ~cpof indium) and 5 ml of a solution containing 6 mg of interfering ion were mixed. Solid potassium iodide (3.735 g) and IOM sulphuric acid (3 ml) were added. to make the solution I.5M in potassium iodide and X4 in sulphuric acid. in a total volume of I5 ml. The contents of the funnel were mixed to dissolve the potassium iodide. and then extracted for 2 min with a I’!; N-benzylaniline solution in chloroform (two 5 ml portions). The mixture was swirled gently and the chloroform layer separated. The organic phases were combined and indium stripped by shaking for 2 min with 2M hydrochloric acid (15 ml). The aqueous layer was transferred to a 25 ml volumetric tlask and concentrated ammonia solution was added dropwise until a turbidity appeared. which w’as cleared with a few drops of 2M hydrochloric acid, and the solution was then diluted to volume with water. A 0.1 ml ahquot of this neutral extract (containing 0.24 /~g of indium) was used for the determination of indium. Derermination of calcium (6-120 pg). Use the procedure for indium, but add 0.1 ml of solution containing 6-120 iig qfcaicium. Plot a calibration curve of “/,I LXcalcium concentration. A new calibration curve should be made daily. Acknowledgement-The
authors thank Professor R. Belcher for his interest and encouragement.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
I I. 12. 13.
14. 15.
G. Siebert, M. Carsiotis and G. W. E. Plant, J. Bio/. Chem., 1957.226, 977. A. Kornbergand W. E. Pricer, Jr., ibid., 1951, 189, 123. E. Adler, H.-von Euler, G. Giinther and M. Plass, Biochem. J., 1939.33. 1028. A. L. Grafflin and S. Gchoa. B&him. Bioohvs. Acta. 1950. 4. 205. J. Moyle and M. Dixon, Biochern. J., 1958, 83, 548. B. Kratochvil. S. L. Boyer and G. P. Hicks. Anal. Chem., 1967,39,45. P. Baum and R. Czok, Biochem. 2.. 1959,332, 121. W. J. Blaedel and G. P. Hicks, Advun. Anul. Chem. Instrum. 1964. 3, 118. A. A. Yadav and S. M. Khopkar. Tulanta, 1971, 18, 833. M. M. L. Khosla and S. P. Rao. Anal. Chirn. Acta., 1972, 58, 389. D. Mealor and A. Townshend, Talanra, 1968, IS, 747 and references therem. A. Gubin, E. Hass. H. Hoffmann and W. Reinmuth. Mitt. cer. Grosskrs.srlhrsit-_rr, F. K. West. P. W. West and F. A. Iddines. Anal. Chan.. 1966.38. 1566. A. Townshend and A. Vaughan, Talon& 1970, 17,299: R. E. Thiers, Meth. B&hem. Anal., 1957,5,273.
1955. No. 33. -135.
EnTymc-catalyscd reactions Zusammenfassung-Die lnhiblerung von lsocitrat-Dehydrogenase durch Blei, lndium und Calcium wurde untersucht. Es werden Methoden skizziert, wie man auf Grund dieser Inhibierung 0,02 -1 hcg Blei und Indium bestimmen kann. Blei kann vor der Enzymreaktion durch Extraktlon mit Tributylphosphat in Isobutylmethylketon von einigen stiirenden lonen abgetrennt werden. Rbsum&On a ttudii I-inhibition de la deshydroginase isocitrique par le plomb, I’indium et le calcmm. On mdique des mithodes pour le dosage de 0.024-I pg de plomb et d’indium. bas&es sur cette inhibition. On peut separer le plomb de quelques ions gsnants par extraction au phosphate de tributyle en lsobutylm6thyldtone. avant application de la rCaction enzymatique.
409
21. pp 401-409
Perpamon Press. 1974 Prmted m Great Brttam
APPLICATIONS OF ENZYME-CATALYSED REACTIONS IN TRACE ANALYSIS-VII* DETERMINATION OF LEAD AND INDIUM BY THEIR INHIBITION OF ISOCITRATE DEHYDROGENASE R. A. SHEIKHand ALAN TOWNSHEND Chemistry Department, The University. P.O. Box 363. Birmingham 15. England (Received
10 Srprernher 1973. Accepred
21 November
1973)
Summary-The Inhibition of isocitrate dehydrogenase by lead. indium and calcmm has been studied. Methods for the determination of 0.024-l pegof lead and indium. based on this inhibition. are outlined. Lead may be separated from some interfering ions by extraction with tributyl phosphate in isobutyl methyl ketone. before application of the enzymic reaction.
Isocitrate dehydrogenase [isocitrate : NADP oxidoreductase (decarboxylating), EC. 1.1.1.42], ICDH, catalyses the oxjdative decarboxylation of isocitrate to a-ketoglutarate. A nicotinamide nucleotide is the co-enzyme used as the oxidant and a doubly-charged cation such as Mg2+ or Mn’+ 1srequired to activate the enzyme: CH,COOH
CH,COOH
I
I
HC-COOH
+ NADP+
Mn”,ICDH
HCH
\
I
I
+
NADPH
+ H+ + CO2
C-COOH
HC-COOH I
/
OH
0 (a-ketoglutaric
(isocitric acid)
acid)
The reaction proceeds from isocitrate to x-ketoglutarate through an enzyme-bound oxalosuccinate intermediate.’ There are two isocitrate dehydrogenases.2 One requires NADP as its co-enzyme whereas the other requires NAD. The enzyme used in the present study is the purified NADP-linked enzyme, obtained from pig’s heart.3-” molecular weight 61 x 103. It is inhibited by a wide range of metal ions.(’ and is activated by manganese(H). magnesium, cobalt(II) and zinc ions.(’ The enzyme has been used to determine klg amounts of magnesium in blood serum? and ng amounts of manganese, ’ by means of their activation property. An interesting method of titrating a sequence ofmetal ions (including Mg’+. Mn’+, Zn’+. Co’+, Cu’+, Cd’+ and Ni2’) with EDTA. using the activation and inhibition effects to follow the progress of the titration. has also been developed, using this enzyme.6 Earlier workers showed that ICDH was inhibited by ng amounts of a number of metal ions. This paper reports an investigation of the inhibitory effects of lead, indium and calcium on ICDH. with a view to developing sensitive methods for the determination of these * Part VI’ Thrm
1970. 17, 299 401
402
R. A.
SHEIKHand ALAN TOWNSHEND
elements. As many other ions would interfere in such determinations, either by inhibiting or activating the enzyme, attempts are made to utilize chemical separation techniques to improve selectivity. In the discussion that follows, all metal ion concentrations refer to the final solution in which the enzyme-catalysed reaction takes place, unless otherwise indicated. o,so 4
f 0
;
.cp
0.45
8 e, $ 8
0.40
I
I
I
I
5
IO
I5
20
Incubation
time,
mm
Fig. 1. Effect of incubation time of ICDH with (A) 20 ng of PbzC per ml in pH 83 tris buffer and (B) with 20 ng of In3 * per ml in unbuffered water.
INVESTIGATION
Efict
OF
EXPERIMENTAL
CONDITIONS
of manganese(IZ) ions and pH on the activity of ICDH
NADP-linked isocitrate dehydrogenase is active only in the presence of an activating metal ion. It is most effectively activated by manganese(I1) ion.8 and these were used in the present investigation. It was necessary to establish the manganese(I1) concentration and contact time with the enzyme required to induce maximal enzyme activity, under the conditions used for the metal ion determinations. On incubation of the enzyme with 6 x lo- 5 M manganese(I1) at 0” in 0.05M Tris buffer (pH 7.5) or water, the activity increases slightly at first but there is no further increase in activity after 10 min. As maximal inhibition of the enzyme by lead or indium is achieved after 15 min incubation of inhibitor with ICDH (Fig. l), this incubation time was used for both activator and inhibitor in all subsequent experiments. The effect of manganese(I1) concentration on enzyme activity, after incubation in pH 75 Tris buffer solution for 15 min, is shown in Fig. 2. Under these conditions, which are those recommended for the determination of indium (see below), greatest enzyme activity is stimulated by lo- ‘M manganese(I1). The same concentration of manganese(I1) was found to be necessary to give maximal activation under the conditions recommended for the determination of lead (pH 8.5 Tris buffer, cu. 0.13 units of enzyme per ml of reaction mixture). The change in enzyme activity with pH (Fig. 3) shows that maximal activity occurs at pH 7.4.
Enzyme-catalysed
o.44
403
4.5
$0
5.5
Fig. 2. ElTect of Mn2+ concentration
reactions
on ICDH activity after 15 min incubation in pH 7.5 Tris buffer.
Inhibition by lead As indicated above, inhibition by lead is maximal after 15 min at O”, and is extensive even after 1 min. Incubation is best done at o”, because the enzyme slowly loses activity at 25”. Figure 3 shows that inhibition is greatest at pH 8.5. Thus, combination of these conditions gives the greatest sensitivity to lead reproducible inhibition being obtained over the range 2-70 ng of lead per ml (Fig. 4). Kratochvil et aL6 have reported that NADP-linked ICDH is inhibited by traces of many metal ions. The effect on enzyme activity of incubating the enzyme with lead (20 ng/ml) and other ions (2 pg/ml) in pH 8.5 OOSM Tris buffer was studied. The results are summarized in Table 1. All the ions investigated interfered, by inhibition (high results) or activation I
.o-
0.75J s 0 5 a
0.51
I
I
I
7.0
e.0
I
I 9.0
PH
Fig. 3. Effect of pH on (a) ICDH activity; (b) inhibition by 40 ng of In”+ per ml; (c) inhibition by 20 ng of Pb2+ per ml.
404
R. A.
SHEIKH
and
ALAN TOWNSHEND
Calcium concentration,
Indium
fig
or lead concentration,
/ml
q/ml
Fig. 4. Inhibition of ICDH by various concentrations of (a) Pb”. (h) Cal’. under the recommended conditions, and of It?+ after incubation in (c) unbuffered water: (d) pH 75 Tris buffer solution.
(low results) except for Al 3+ . Complete inhibition of the enzyme was observed in the presence of Hg ‘+, Ag+, Zn2+, Cu2+ and Cd 2+ . Most of these results are in agreement with the effects noted previously.6 In an attempt to develop a selective method for lead, the extraction method of Yadav and Khopkarg was investigated. Tributyl phosphate (TBP, 30% solution in isobutyl methyl ketone) is reported to separate lead selectively from a wide range of metal ions’ in a medium that is 3M in hydrochloric acid and 2M in lithium chloride. The most probable composition of the extractable lead species is PbC12*2TBP.’ Lead can readily be stripped from the organic phase with water, for enzymic determination. The final aqueous extracts were flushed with a slow stream of nitrogen to get rid of the last traces of isobutyl methyl ketone, which was found to inhibit the enzyme. With use of this method lead (45 /lg) could be determined in the presence of up to 4.5 mg of Bi3+, &I+. and Cal+. 2.25 mg of Hg2+. and 1.1 mg of Ag+. These are the maximum tolerance limits as reported by Yadav and Khopkar,9 and greater ratios were not investigated. The results are shown in Table 2. Itlhihition by in&m Conditions for maximal inhibition of ICDH by indium were found in a similar manner to those for lead. With incubation in unbuffered water for at least 15 min. and the enzyme reaction carried out at pH 7.5. fairly reproducible inhibition was achieved over the range Table I. Direct determination of lead (20 q/ml) m the presence of other ions (2 jcgiml)
Enzyme-catalysed Table 2. Determmation
Other cations. (pg.irnl) Pb’+ found, ng/rnl
reactions
405
of lead after extraction with 30% tributyl phosphate in isobutyl methyl ketone (Pb*’ taken = 15 n&ml) 15
Bi3’ (1.6) 13 16
Ba2+ (1.6) 16 13
Ca’+ (1.6) 13 16
Hg’+ (0.8) 13 16
Ag’ (0.4) 16 13
2-l 00 ng of indium per ml (Fig. 4). Results for the determination of indium in pure solution are shown in Table 3. When the enzyme was incubated with indium in pH 7.5 buffer solution for 15 min, the inhibition was slightly less sensitive (Fig. 4) allowing 3-100 ng indium per ml to be determined. Determinations were more reproducible, however (Table 3). In order to eliminate the interference of other metal ions, an attempt was made to determine indium after solvent extraction separation. The procedure of Khosla and Rae” using extraction by N-benzylaniline in chloroform from a hydriodic acid medium was investigated. The extraction procedure is simple and the separation of the organic and aqueous phases is very rapid. Indium is later stripped from the organic phase with 2M hydrochloric acid. When a 9”/, w/v solution of N-benzylaniline in chloroform, reported by Khosla and Rae” to separate indium from most metal ions, was used, some benzylaniline appeared in the final aqueous solution, resulting in the complete inactivation of the enzyme. To overcome this problem the effect of smaller benzylaniline concentrations was investigated. It was found that the concentration had to be decreased to 1% to prevent inhibition by the extractant. However, when a lOO-fold amount (by weight) of some other ions was present with 60 pg of indium. and the 1% extractant solution was used, high results for indium were obtained (Table 4). Complete inhibition was observed after extraction when mercury and cadmium ions were present in lOO-fold amount.
The incubation of the enzyme with calcium was done in unbuffered water for 15 min. The pH of the reaction solution was the same as for the determination of indium. The method allowed the determination of 0.5-10 pg calcium per ml in the absence of interfering ions (Fig. 4). Stahilitj* of‘ the enl!rtle-ilzhihitor complexes
The reaction between a cationic inhibitor (M”+) and an active site of an enzyme, (E), can be represented by the equation E + rrMX+= M,“+. E
(1)
where II is the number of metal ions bound per active site.” Table 3. Determination No huff27
O.OSM
7-m
bufl?r. pH 7 5
of indium
Taken, rlg/rvl 2 Found. ng/rnl 3
5 7
10 13
30 36
80 85
Taken. ng/rn/ Found. ng/rnl
10 11
20 23
40 40
60 61
80 77
100 100
R.
406
Table 4. Determination Other cations In3 + found ng/mf
of
A.
SHEIKH and ALAN TOWNSHENII
indium after extraction with 194 N-benzylaniline ng/ml; other ions = 2 fig/ml) _ 19
20
Pbl+ 47
ca” 89
Co”+ 47
Al3f 39
in chloroform. (Indium taken = 20
&2+ 89
Ni” 58
Mg2’ 28
&‘f 39
Cd”+ H$+ >200 3200
An equilibrium constant, R, is given by K = CUW’I” CM;+ . E]
(2)
The relative activity, RA, is proportional to the concentration of free enzyme sites, [El. Therefore. the ~on~ntration of inhibited enzyme, CM;’ . EJ, is proportionai to (I- RA) so 1-RA _ CM:+ .E] RA CEj
(3)
where RA is the fractional enzyme activity relative to that of the uninhibited enzyme under the same conditions. A combination of equations (2) and (3) gives
1-RA _ CM”‘]” RA
l-RA
log RA
K
= n log CM”‘] - log K
t51
A plot of log (1 -RA)/RA us. log CM”‘] should therefore give a straight line of slope n and intercept log K. The results of such plots for lead, indium and calcium are summarized in Table 5. All three plots give straight lines and the values of n are all approximately unity, indicting that one ion per active site is responsible for the inhibition. The equilibrium constants confirm that the strength of binding by the enzyme increases in the order Ca2+ Q In3+ < Pb2+. Table 5. Equilibrium constants for the enzyme-inhibitor
Inhibitor In3 +
Incubation, PH 7.5 HtO*
I1
complexes
K,ll?Ok/l.
I.15 I.20 091 0.96 @90
64 6.0 3.4 4.3 24
x x x X x
lo-’ lo-’ to-’ lo-’ lo-’
Pb’ +
8.5
1.04 l-16 1.20 0.94 1.04
1.1 X I.3 x I.3 x 1.5 X 1.4 x
ca2+
H1O*
I .07
I.2 X 10-d
* II nhullrcd.
to-7 lo-’ 1o-7 lo-’ IO-’
Enzyme-catalysed reactions
407
Adsorption of metal ions on glassware
Traces of metals are readily adsorbed by glass surfaces. 12*13*I4 During these investigations. it was confirmed that lead is adsorbed by glass, which could lead to serious errors when ng amounts of this metal ion were being measured. This source of error could be
eliminated, however, by putting the enzyme solution into the incubation flask first, followed by the lead solution. The lead was preferentially bound by the enzyme, and no significant amount was lost on the glass surface. To minimize any possible contamination from adsorbed lead or other metal ions, however, all glassware was soaked before use for several hours in a saturated aqueous solution of disodium EDTA, and rinsed thoroughly with water. Adsorption was also minimized by leaving metal ion solutions in contact with the glass for as short a time as possible. As polyethylene is not so susceptible to these adsorption effects,” all stock metal ion solutions were stored in polyethylene bottles.
The methods described allow the reproducible determination of ng amounts of indium and lead and rather larger amounts of calcium. As the enzyme studied is also inhibited by a wide range of other metal ions, means of prior separation of indium and lead from potential interferences were sought. Solvent extraction was successfully applied for the separation of lead, provided no significant amount of extractant found its way into the presence of the enzyme. Isobutyl methyl ketone and N-benzylaniline both inhibited ICDH, but whereas the ketone could readily be eliminated from the final aqueous solution, and thus did not interfere in the determination of lead, N-benzylaniline could not be prevented from finding its way into the final aqueous solution, if its concentration was to be sufficient to separate indium from other metals. EXPERIMENTAL The enzymic reactions were momtored m IO mm silica cuvettes in a Beckman DB spectrophotometer suring at 340 nm the rate of formation of NADPH at constant temperature (25.0 k @lo).
by mea-
Rragmts
Water distilled from an all-glass apparatus was used throughout. En-_~~ne.Highly purified pig’s heart isocitrate dehydrogenase in 50% glycerol (Sigma Chemical Co.) of activity 5 fitmole. mg- ‘. min- ’ was stored at 4”. A standard solution was prepared from this stock enzyme daily before use by diluting. with 0.05M Tris buffer of the required pH, 20-fold v/v for indium and calcium determinations (pH 7 5) and lo-fold for lead determinations (pH 8.5). The standard enzyme solutions were kept in an ice-bath during use. T~rs(h~dro.~~r~~~t/~~~)~~~~t/~~~at~~~t~~ (Tris) bufir solution, DOSM. Tris (3.03 g) was dissolved in about 400 ml of water. adjusted to the required pH with 0.1&f analytical-grade hydrochloric acid and diluted to exactly 500 ml wnh water The solution was kept m a thermostat at 25” before use. Substrates. A reaction mixture consisting of 30 mg of analytical-grade,trisodium or_-isocitrate dihydrate (ICA) and I7 mg of the monosodium salt of nicotinamide adenine dinucleotide phosphate (Sigma Chemical Co.) in 25 ml of 0.05M Tris buffer solution of the required pH was used. This mixture was kept in an ice-bath during use and kept frozen when not in use. When frozen it was stable for weeks. Mrtal ion solutions. Stock solutions of indium and lead (200 pg/ml), calcium (120 &ml) manganese (5.0 x 10-‘M) and possible interfering ions (ca. 1 mg/ml) were prepared from analytical-grade reagents and stored m polyethylene bottles. More dilute solutions were prepared immediately before use by appropriate dilution of the stock solutions. Procedures D~~tcwmotrort ofleud (24-840 y). To a 2 ml volumetric flask add Tris buffer (pH 8.5. ca. 1.0 ml) and exactly 0.1 ml (0.1 mg of protem = 0.5 units) of enzyme solution previously cooled m ice. Mix gently and add
408
R. A.
S&H
and
ALAN TOWNSHEXD
I.2 x IO-‘M manganese solution (exactly 0.1 ml). Mix the contents of the flask aeain and add ~0.5 ml of lead solution, at O”,containing 24-840 ng of lead. Make up to the mark with pH 8.5 T& buffer at 0-. trux thoroughly and place in ice for 15 min. Add exactly 0.5 ml of this solution to a mixture of ICA-NADP in pH 85 Tris buffer (05 ml), pH 8.5 Tris buffer (1.0 ml) and water (1-Oml) all at 25.0”. After exactly 15 min at this temperature. measure the absorbance (Ai) at 340 nm in a 10 mm cuvette against water. Repeat the experiment in the absence of the inhibitor to obtain the absorbance (A,) resulting from the uninhibited reaction and calculate the percentage inhibition (%I = IOO(A,,,-A#&). Plot a calibration curve of ‘41 us. lead concentration for the results obtamed from standard lead solutions taken through the procedure. A new calibration curve should be made daily. Liquid-liquid extraction of lead with triburylphosphare. This was investigated as follows. To 1 ml of solution containing 45 pg of lead were added (4 ml of solution containing < 4.8 mg of Bi”‘. Ba” or Ca”. 7.4 mg of Hg’+ or I.2 mg of Ag+). To this solution, in a 100 ml separating-funnel. I2M hydrochloric acid (5 ml) and 4M lithium chloride (10 ml) were added, to make their respective concentrations 3M and 2:M In a total volume of 20 ml. The mixture was extracted with 10 ml of 30’4 tributyl phosphate in isobutyl methyl ketone b! shaking briskly for 5 min. The layers were allowed to separate, the aqueous layer carefully removed. and lead stripped from the organic phase by shaking with water (two 10 ml portions). The aqueous lead solutions were quantitatively transferred to a 25 ml volumetric flask and made up to volume with water. Isobutyl methyl ketone was removed from the aqueous solution by passage of a slow stream of nitrogen for 15 min. Aliquots of this aqueous solution (0.1 ml contained 0.18 pg of lead) were used for the enzymic determination of lead. Determinarion ofindium (24-1200 ng). Use the procedure described for lead, but incubate the enzyme (0.1 ml containing 50 pg of protein = 0.25 units) in water instead of tris buffer solution and add < 0.5 ml of solution containing 24-1200 ng of indium. Carry out the enzyme-catalysed reaction in pH 7.5 Trls bufier solution at 25.0 f 0 1”.Plot a calibration graph of%1 us. indiumconcentration. A new calibration curve should be made daily. Separation of indium by solvent extracrion with N-benzyluniline. The investigation was made as follows. In a 50 ml separatory funnel distilled water (6 ml). indium solution (I ml containing 60 ~cpof indium) and 5 ml of a solution containing 6 mg of interfering ion were mixed. Solid potassium iodide (3.735 g) and IOM sulphuric acid (3 ml) were added. to make the solution I.5M in potassium iodide and X4 in sulphuric acid. in a total volume of I5 ml. The contents of the funnel were mixed to dissolve the potassium iodide. and then extracted for 2 min with a I’!; N-benzylaniline solution in chloroform (two 5 ml portions). The mixture was swirled gently and the chloroform layer separated. The organic phases were combined and indium stripped by shaking for 2 min with 2M hydrochloric acid (15 ml). The aqueous layer was transferred to a 25 ml volumetric tlask and concentrated ammonia solution was added dropwise until a turbidity appeared. which w’as cleared with a few drops of 2M hydrochloric acid, and the solution was then diluted to volume with water. A 0.1 ml ahquot of this neutral extract (containing 0.24 /~g of indium) was used for the determination of indium. Derermination of calcium (6-120 pg). Use the procedure for indium, but add 0.1 ml of solution containing 6-120 iig qfcaicium. Plot a calibration curve of “/,I LXcalcium concentration. A new calibration curve should be made daily. Acknowledgement-The
authors thank Professor R. Belcher for his interest and encouragement.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
I I. 12. 13.
14. 15.
G. Siebert, M. Carsiotis and G. W. E. Plant, J. Bio/. Chem., 1957.226, 977. A. Kornbergand W. E. Pricer, Jr., ibid., 1951, 189, 123. E. Adler, H.-von Euler, G. Giinther and M. Plass, Biochem. J., 1939.33. 1028. A. L. Grafflin and S. Gchoa. B&him. Bioohvs. Acta. 1950. 4. 205. J. Moyle and M. Dixon, Biochern. J., 1958, 83, 548. B. Kratochvil. S. L. Boyer and G. P. Hicks. Anal. Chem., 1967,39,45. P. Baum and R. Czok, Biochem. 2.. 1959,332, 121. W. J. Blaedel and G. P. Hicks, Advun. Anul. Chem. Instrum. 1964. 3, 118. A. A. Yadav and S. M. Khopkar. Tulanta, 1971, 18, 833. M. M. L. Khosla and S. P. Rao. Anal. Chirn. Acta., 1972, 58, 389. D. Mealor and A. Townshend, Talanra, 1968, IS, 747 and references therem. A. Gubin, E. Hass. H. Hoffmann and W. Reinmuth. Mitt. cer. Grosskrs.srlhrsit-_rr, F. K. West. P. W. West and F. A. Iddines. Anal. Chan.. 1966.38. 1566. A. Townshend and A. Vaughan, Talon& 1970, 17,299: R. E. Thiers, Meth. B&hem. Anal., 1957,5,273.
1955. No. 33. -135.
EnTymc-catalyscd reactions Zusammenfassung-Die lnhiblerung von lsocitrat-Dehydrogenase durch Blei, lndium und Calcium wurde untersucht. Es werden Methoden skizziert, wie man auf Grund dieser Inhibierung 0,02 -1 hcg Blei und Indium bestimmen kann. Blei kann vor der Enzymreaktion durch Extraktlon mit Tributylphosphat in Isobutylmethylketon von einigen stiirenden lonen abgetrennt werden. Rbsum&On a ttudii I-inhibition de la deshydroginase isocitrique par le plomb, I’indium et le calcmm. On mdique des mithodes pour le dosage de 0.024-I pg de plomb et d’indium. bas&es sur cette inhibition. On peut separer le plomb de quelques ions gsnants par extraction au phosphate de tributyle en lsobutylm6thyldtone. avant application de la rCaction enzymatique.
409