Enzymatic determination of zinc below one part per billion

Atdgrica

Chirnicu Aclo, 70 ( 1974) 85-93 Publishing Company.

‘c Elscvicr Scicntilic

ENZYMATIC BILLION*

PAVEL

LEHKY

Departmwr

(Rcceivcd

DETERMINATION

85 Amsterdum

OF

- Printed

ZINC

in The Ncthcrlands

BELOW

ONE

PART

PER

und ERIC A. STEIN

oJ’ Biochernisrry.

3rd October

Uuiuersiry

0-f Gerworr (Switzerlurld)

1973)

Particulate aminopeptidase from pig kidney’ (EC 3.4.1.2) is a metalloenzyme that contains 2 g-atoms of zinc per mole (m.w. 280,000)2. Although tightly bound, the metal can be removed from the enzyme and the resulting metal-free inactive apoenzyme can be prepared in gram amounts 3. This apoenzyme has a high affinity for zinc and is readily reactivated by the latter metal, even after extended periods of storage, or upon exposure to heat and extreme pH. The assay of aminopeptidase activity is easy to perfqrm, fast and sensitive, and does not require a more complex instrument than, a recording photometer. As the recovery of activity is strictly proportional to the amount of zinc added to the apoenzyme (up to saturation), the aminopeptidase assay allows the quantitative determination of zinc(I1). These features single out aminopeptidase as a reagent ideally suited for the assay of very low concentrations of zinc(I1): a sample containing as little as 5 pg Zn ml-’ will cause a significant reactivation of aminopeptidase. The analytical procedure described here is not only highly sensitive, but also specific, since 0.1 ng Zn ml-’ can be assayed with lo”/, accuracy even in the presence of a ten- to hundred-fold excess of most other metals. EXPERIMENTAL

“Specpure” divalent salts of Mg, Mn, Ca, Co, Ni, Cu, Zn, Cd, Hg (Johnson Matthey Chemicals, London), TES (N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid: Sigma Chemical* Company), Chelex X-100 chelating resin (Calbiochem), and Suprapure hydrochloric acid and EDTA (Merck) were used. All other chemicals were of analytical grade. Demineralized water was double-distilled in an all-quartz apparatus. The still was rinsed daily to prevent accumulation of metal ions. Double-distilled water was stored in 30-l metal-free polyethylene bottles for no longer than one week. The zinc content of the water varied from 5 to 30 pg ml- 1 as determined by the enzymatic procedure described below. Containers and pipettes Glassware was avoided * This

paper is dcdicatcd

in order

to Professor

to minimize

F. Lcuthurdt,

Zurich,

contamination on the occasion

by traces

of

of his 70th birthday.

P. LEHKY.

86

E. STEIN

metals. Only polyethylene or polypropylene vessels were used; they were filled with 1 M hydrochloric acid to remove metal contaminants. After 24 h, the containers were carefully emptied, rinsed with water, capped and stored in a dust-proof cabinet. Micropipettes (“BB*’ constriction type, Calbiochem. made of polymethylpentene) were cleaned with water, 5O”/0HN03 (1~0 nnalysi), water, 1 M HCl (Suprapure) and again with water. In most cases, solutions were made or diluted by weighing. Prepara

tio!l oJ‘ metal-free TES buf.‘fer TES solutions. pH 7.0, were passed through a 1.2 x 17-cm polyethylene filled with Chelex freshly rinsed with 250 ml of water and stored. Before buffer was filtered once more through Chelex.

column use, the

Aminopeptidase was isolated in pure form (specific activity 27-30 I.U.) from was prepared by removal pig kidney* as described previously 2. The apoenzyme of zinc as follows: 10 ml of enzyme solution (5-10 mg ml-’ in 0.05 M TES, pH 7.0) was treated with a 3 volume of wet Chelex; after 12 h at 4”, the resin was centrifuged off and washed with an equal volume of metal-free TES buffer. The combined washings and enzyme solution were treated twice more with Chelex. At this point the protein concentration was 2-3 mg ml- I, as determined from (Perkin-Elmer the absorptivity3 E fF”= 16.9. Metal analysis by atomic absorption Model 303 spectrophbtometer fitted with a recorder) indicated that the zinc content was below 0.02 p.p.m., the detection limit; the enzymatic activity was approximately 0.50/, of that of untreated zinc-containing aminopeptidase. Such a solution of apofor 6 months, suffers no deterioration and regains enzyme, stored frozen at -20” full activity upon addition of zinc. When inactive apoaminopeptidase is added to the zinc-containing sample that is to be analyzed, a stoichiometric amount of enzyme reverts within a few seconds to its active form. To determine how much apoaminopeptidase has been reactivated, an aliquot is withdrawn from the reaction mixture and assayed for activity. The correlation between zinc content and enzyme activity yields a straight line between 0 and 2 g-atoms of zinc per mole of aminopeptidase (280,000), corresponding to 0 and lOO”/0enzymatic activity, respectiveiy3. The latter value is essentially constant for a given preparation of aminopeptidase, owing to the great stability of the enzyme. (A neutral aminopeptidase solution (0.5 mg ml-‘) stored for 2 years in a sealed tube, at room temperature, was shown to retain 95% of its original activity.) Moreover, excess of zinc has no effect on the enzymatic activity up to 200 g-atom mol- ‘. Therefore, this assay yields all the information necessary to calculate the amount of zinc present in the sample, at least in the absence of interfering substances. When zinc has to be determined in the presence of other cations, or interfering substances of any kind, an internal standard should be used (see below). * The same MO.. USA.

enzyme

can

now

bc obtained

commercially from Sigma Chemical

Co.. St. Louis.

.

ENZYMATIC

DETERMINATION

87

OF ZINC

After determination of the extent of reactivation by a sample of unknown chemical composition, one should check that nothing has interfered with the potential activity of apoaminopeptidase during interaction with the sample. To this effect, the apoeniyme is saturated with excess of zinc. If the reconstituted aminopeptidase shows a specific activity different from that of the native enzyme, inhibition or denaturation of aminopeptidase must be suspected and the zinc determination considered as invalid. However, this discrepancy seldom occurs (see Discussion). Determimltion of amiuopeptidase Nctivity The substrate used is 16.6 mM L-leucine-p-nitroanilide (first dissolved in 10% ethanol) made 0.5 M in TES and 0.1 mM in EDTA. The latter metal-binding agent is necessary to “neutralize” traces of metals that may contaminate the substrate3. The rate of liberation of p-nitroaniline is followed for 5-10 min at 405 nm with a recording photometer (.s505 = 9620 1 mol-’ cm-’ ). The assay is performed at 37” and pH 7.0 in a l-ml glass cuvette with a l-cm light path, by adding 0.5 ml of apoenzyme-sample mixture to 50 /cl of EDTA-containing substrate. This order of addition is imperative to prevent adventitious enzyme activation by cuvette-derived metal ions. Zinc

determination Depending upon whether the expected zinc concentration is in the 5-50 pg ml-’ or the 25-250 pg ml-’ range, one adds either 10 or 50 ~1 of apoaminopeptidase solution (0.1 mg ml-r) to a sample diluted to 10 ml with metal-free buffer in a lo-ml polyethylene bottle. The latter is thermostatted at 37”. temperature equilibrium taking about 10 min. A 0.5-ml aliquot is added to 50 it1 of substrate in the photometer cell and the aminopeptidase activity A,, is determined. After the increase in absorbance has been recorded For l-5 min. 3 itI of 2 mM zinc sulfate (2 ,uM excess over EDTA) are added to the cuvette in which AZ,, has just been measured; this results in an immediate increase in activity (A?). The latter measurement, made to ascertain that the apoenzyme has not been porsoned, can be omitted when one is working with a “clean” system, as A, can be calculated from the amount of apoenzyme used for the assay. The percentage reactivation P, is given by the relation P, =(A=” - A,,). 100/A,, where At, is the blank. The latter is determined as is done Fdr AZ,, but Gn the absence of sample. To demonstrate that this analytical procedure applies to very low zinc concentrations, and that the relation between extent of reactivation and zinc concentration is linear, two calibration curves were established. The plots depicted in Fig. 1 and 2 were fitted by a computer program based on linear regression. The 95% confidence limits for each point are indicated by bars. The correlation coefficients calculated from the data of Fig. 1 and 2 were 0.991 and 0.995, respectively. For both sets of data, the slope of the curve was significantly different from zero. As the deviation From the origin was statistically not significant, there is no reason to suspect any systematic source of error in the measurements, Determination of copper, cobalt and nickel Most metals other than zinc are unable

to confer

activity

to the apoenzyme.

P. LEHKY,

c

.o

E. STEIN

60

2

z 40

E a s

20

0

0

10

20

30

II

40

50 pg Zn

Fig. 1. Calibration

curve for zinc at conccntrotions

ml-’

of 5-50 pg ml-‘.

!

pg Zn ml-’

Fig. 2. Calibration

curve for zinc at concentrations

of 25-250

pi ml- *.

The following cations, Be’+, Mg’+, A13+, Ca2+, Sc3+, Cr3+, Mn2+, Fe’+, Fe3+, Sr2+, Pd2+, Agf, Cd’+, Sn2+, Bazf, La3+, Hg2+ and Pb2+, were investigated, and failed to bring about any restoration of activity. In contrast, Cu’+, Co’+ and and therefore this enzyme could also Nit+ could reactivate apoaminopeptidase3 be used to estimate the latter cations. Because of the much lower affinity of the protein for Cult, Co2+ and Ni2+ than for Zn2+, the method in this case is less sensitive by two orders of magnitude. It is also less selective, as each of the three cations can be assayed only in the absence of the other two. In contrast to the reaction catalyzed by the zinc enzyme, which proceeds linearly with time, Fig. 3 shows that the rate of the reaction catalyzed by the Cu-, Co- and Ni-enzyme levels off within lo-30 min. Apparently, Cut+, Co2+ and Ni2+ are progressively removed from aminopeptidase during the assay, which is performed with EDTA-containing substrate”. Thus, EDTA, which does not affect the zinc enzyme under the conditions of the assay3, provides a means of distinguishing Cu2 +, Co2 + and Ni2 + from Zn2 +, besides masking any metal contaminants in the substrate.

ENZYMATIC

DETERMINATION

OF ZINC

89

E c 8

a? Time

(mln

1

Fig. 3. Hydrolysis of leucine-p-nitroanilide by Zn-. Co-, Ni- and Cu-aminopeptidasc in the presence of EDTA. The peptidases were prepared by reactivating the apocnzymc in the absence of EDTA with a two-fold excess of various metals. The reaction was started by the addition of the enzyme to the substrate. The tinal enzyme conccntrntion was 0.75 pM.

The procedure for the determination of Cu2+, Co2+ and Ni2+ is the sar& as for zinc, except that a higher concentration of apoenzyme (50 pg in 10 ml of buffered sample) must be used as the assay system contains much more metal than is the case for zinc. For determination of the aminopeptidase activity, the substrate is diluted 10 times with water; the assay is started by addition of 50 ,~l of apoenzyme-sample mixture to 0.5 ml of diluted substrate. Figure 3 shows clearly that it is imperative to measure the initial reaction rates. The calibration curves for Cu2+, Co’+ and Ni2+ at concentrations ranging from 0.25-2.5 ng ml -I are shown in Fig. 4. Deterrninatiorz of zirtc in the presence of other metals To demonstrate that the enzymatic estimation of zinc is possible in the presence of a wide array of other cations, a typical sample for analysis was made up, containing 100 pg Zn ml-’ and a lo- to lOO-fold molar amount of another cation, in TES buffer. After addition of 50 ,~l of a 0.1 mg ml-’ apoenzyme solution 200 -

cu ,,...3’ .,..‘,

/+‘. / ,.&/” co ,,,...”0.. ,,...’..’ ,.n....” ,,...’ _.....,’ ...” ..D”’ 0(0’....J:::,8 ,.,.,......,

150.-g ;ii ., f; lookz (I 9 0

../.$’ .,@.....“’

50-

./‘;; ,..A ..‘:(.(.. ,...&: .. ,/::,... o::::.. ,,,,:,::a ...*

,,.....,..,... s...‘.““” /’ /. ,~, ,,,,,,~ ,,.,,.,,....... ...((.,“‘..‘. .....~.. ,,.... ..,,,,,,.,......‘......,..‘. . . . 0

.,. ,,......

‘..

0.0

Fig. 4. Calibration

,

0.5

curves

I

f

1.0

for copper,

,,.,,,, ,.,,.....l ,.,.,,,N’!‘ib

1.5

2.0

2.5

ng cobalt

and nickel.

Me

ml-’

90

P. LEHKY,

E. STEIN

to 10 ml of buffered sample, an aliquot of 0.5 ml was withdrawn and the extent of reactivation determined. In a second step, the zinc concentration of the remaining 9.5 ml of apoenzyme-sample mixture was increased by adding 111 pg Zn ml-’ as an internal standard, and the mixture reassayed. The resulting increase in activity was used to calculate the zinc concentration of the original sample (100 pg ml- ‘). Table I presents the results of a series of such experiments performed in presence of a large variety of cations. and shows that zinc can be determined with a IOo/, error in the presence of a lOO-fold molar amount of most metals. or of a 25-fold amount in the case of iron, cadmium, mercury or lead. The least favorable situation arises with cobalt, copper and nickel, where the loo/, error level is already reached in presence of a IO-fold molar amount of the cation. As shown in Fig. 3, it is important in this case to let the reaction proceed for lo-30 min until the reaction rate becomes linear (as a consequence of the chelation of Cu2+, Co2+ and Ni*+) before Az, is determined. TABLE

1

DETERMINATION

OF ZINC

IN THE

PRESENCE

(The ;~vcrages of three scparatc dctcrminations standard deviations: the cxpcrimenktl conditions Molur

f~~cirIti tni- ‘)

of rtwrtri iou

21, (py

NH,’ BC2+ Mg2+ A13+ cE12+ scJ+ Cr3 + MnZ+ Fez+ co2 +

100+4 94_el 1.03 * I I IO1 *8 95+5 106-1-7 103+3 9824 90* I lOI&-

ufnf~w11

100x 100 x 100x 100 x 100x 100 x 100x 100x 25 x 10 x

g2++ Pd=+ Ag+ Cd2+ $1 Hg2* Pb2+

10x 10x 100x 100x 100 x 2s x 100% 100 x 25 x 25 x

CATIONS

of 100 pg Zn ml-’ as ZnS04 arc dctailcd in the text.) ZfI (pg

Ni”

OF OTHER

are presented

with

fiJw1ti Iv/-‘)

128&S 102+7 98&8 109+3 102c4 103* IO 97&8 1os+3 96+9 93&5

Determination of zinc in ph ysiologicul fluids As an example, the enzymatic determination of zinc was cairied out on 2 ~1 of bovine serum. The average of 6 measurements gave the value 1.06 pg Zn ml-’ with a standard deviation of kO.03. Two determinations by atomic absorption spectrometry on a 300-fold .larger sample gave 1 09 and 1.10 /lg Zn ml- ‘. DISCUSSION

During the last decade, various enzymes have been proposed as highly sensitive reagents for the determination of trace metals. Guilbault4 has provided an extensive review of the literature. However, enzymatic analysis has turned out to be generally disappointing because many metals proved to interfere with the assay, either by activating or by inhibiting the enzyme. Activation of an enzyme by an extrinsic metal activator is not selective enough to make such a system attractive

ENZYMATIC

DETERMINATION

OF ZINC

91

from an analytical point of view. The desirable selectivity may be achieved only in the instance of a genuine metalloenzyme, when a given metal is incorporated within the folds of the polypeptide backbone of the enzyme, in a unique structure that is so tight that the metal is considered to be part of the enzyme molecule’. . The -pioneering work of Townshend and Vaughan on the enzymatic determination of zinc’*” deserves mention with respect to such enzymes. Pig kidney particulate aminopeptidase has been shown to be a suitable metalloenzyme containing 2 g-atom of zinc per mole. Although zinc is very firmly bound, a metal-free, inactive form of the enzyrhe (apoaminopeptidase) can be obtained3. In the presence of traces of free zinc ions, the apoaminopeptidase molecule reverts to a powerful catalyst able to hydrolyze lo4 molecules of substrate per min. mole of substrate can be readily estimated, aminoAs the hydrolysis of lo-’ peptidase (and consequently zinc) can be assayed with extreme sensitivity. Under ideal conditions, the procedure described above might allow the determination of zinc at concentrations lower than 1 pg ml- ‘. In practice, however, the sensitivity of the test is limited by the presence of extraneous contaminants. These can be kept at an acceptable level if the precautions described in this work are scrupulously observed. The manipulation involved in the test are limited to: (1) the mixing of sample and ‘reagent, (2) the pipetting of an aliquot, and (3) the mixing with the substrate in the photometer cell. As the latter operation is performed in the presence of EDTA, only the first two manipulations constitute a potential source of contamination. Because of the high stability of the zinc-apoenzyme complex, pH adjustment before the activity determination can often be avoided. In fact, mixing of sample and apoenzyme can be done at any pH between 4.5 and 9.0 for zinc concentrations above 10 ng ml - ’ ; for lower levels, one should not operate below neutrality. The sensitivity of the present method compares very favorably with most other procedures so far described for zinc determinations, for instance, atomic absorptiona, or neutron activation analysisg, and even anodic pulse polarography’O atomic fluorescence flame spectrometry’l, which all require much more complex and expensive instrumentation. The striking stability of apoaminopeptidase represents an important asset in its use as an analytical tool. Stored in solution at room temperature in the presence of toluol, the protein can retain 95% of its potential activity for two years. It is equally resistant to acidic and alkaline conditions, from pH 4 to 11, and is little affected by heavy metals such as Hg2+, Cd2+, Ag+, etc. This behavior, unusual for an enzyme, is probably due to the absence of free sulfhydryl groups and .to the presence of a large proportion of carbohydrates in the enzyme molecule (400 sugar residues, amounting to 20”/0 of the molecular weight)2. A major advantage of the procedure proposed here is that interferences with the determination of zinc are easily evidenced. Non-linearity of the curve reveals the presence of Cu2+, Co2+ or Ni 2+ 3. Failure fully to reactivate the reagent with an excess of zinc (A,,) indicates irreversible poisoning of the reagent. Reversible inhibition may also occur, for instance in the presence of Hg2+, Cd’+, Be’+ or Ni2+. In this instance, one observes a progressive increase of A, to its normal value, as zinc(I1) in excess displaces extraneous cations ‘. In all cases the interferences are obvious and can usually be corrected by means of the “internal standard” method.

92

I’. LEHKY.

E. STEIN

The affinity of apoaminopeptidase for CuZf, Co3+ and NiZ+ is considerably lower than that observed for zinc3, which explains why these metals do not prevent the determination of zinc (at 0.1 ng ml- ‘), even in ten-fold molar amounts. ZnZ+ and either Cu2+ or Co2+ can be assayed simultaneously when they are present in similar concentrations. The initial reaction rate reflects the compounded effects of both zinc and copper (or cobalt), while the rate measured after a further 10 minis due to zinc(lI) only. Simultaneous determination of Zn2+ and Ni2- is less satisfactory. since Ni2+ is not as readily displaced from the reagent as Cu2+ or Co2+. The distinction between free and bound trace elements is of major importance in the investigation of biological samples. By direct determination, classical methods such as atomic absorption allow only the determination of total zinc. In contrast, the enzymatic procedure detects available zinc, i.e. first free zinc, then loosely bound ‘zinc and finally. after denaturation of the sample, firmly ‘bound zinc. This constitutes another asset of aminopeptidase as an analytical reagent. As the level of zinc in most mammalian tissues is ‘significantly higher than that of the “sensitive” cations of nickel, cobalt and copper’ 2, the method described here is particularly well suited to the investigation of such biological samples, and, has been tested successfully in the case of blood serum. Indeed, zinc is the most abundant.intracellular trace element in the human body, and is instrumental in the functioning of many vital metalloenzymes, both intra- and extracellular. A general need for the clinical estimation of zinc levels, comparable to the present interest in iron deficiency, has been predictedi2. This work was supported by grants 4740 and 3.652.71 from the Swiss National Science Foundation. We are grateful to Professor W. Haerdi, from the Department of Analytical Chemistry of this University, for valuable advice, and to M. M. Dumas for his skilful assistance during an undergraduate participation program. SUMMARY

A stable and very sensitive reagent for the determination of zinc is obtained by removing zinc from pig kidney aminopeptidase, a commercially available metalloactivity of the reagent is strictly enzyme. Up to al given limit, the enzymatic proportional to the concentration of zinc ions in the assay system. Aminopeptidase activity is determined by measuring the rate of release of p-nitroaniline from the chromogenic substrate L-leucine-p-nitroanilide. Thus, with a simple recording photometer, rapid and accurate determinations of free zinc ions in concentrations ranging is such from 5 pg-10 pug ml -i can be achieved. The selectivity of the method that 0.1 ng Zn ml-’ can be assayed in the presence of a lo-lOO-fold molar amounts of a wide array of other cations with a relative error below 10%. The analytical procedure has been extended to the assay of Cu2+, Co2+ and Ni2+ in the ng ml-’ range. REFERENCES I E. D. Wachsmuth, I. Fritzc and G. Pficidcrcr, Biochnistr_yq 5 (1966) 169. 2 H. Wncker. P. Lchky. E. H. Fischer and E. A. Stein, He/v. Clliw. Acra, 54 (1971)

473.

-

ENZYMATIC 3 4 5 6 7 8 9 IO

11 12

DETERMINATION

OF ZINC

93

P. Lchky, J. Lisowski, D. Wolf. H. Wuckcr and E. A. Stein. Bioclritt,. Uioph)s. Acrcr. 321 (1973) 274. G. G. Guilbault, EXJVMJ~~~’ Mc~l~otls of Amlysis, Pergamon Press. Oxford, 1970. pp. 176-196. A. Townshcnd and A. Vaughan. Arxrl. Chirrl. Acrrr. 49 ( 1970) 366. A. Townshend and A. Vough:tn. Tdurrrtr. I7 (1970) 289. B. L. Vallcc and W. E. C. Wackcr, in H. Neurath (Ed,). Tllc Profeius, Vol. 5. Academic Press, New York. 2nd cdn., 1970. pp. 61-93. Sroritltrt-tl corrtlirioris Ji)r Arc. in Pcrkin-Elmer’s Atdyrictrl Methotls jiv Atomic Ahsorpriot~ Spectraphororwrr~: Norwalk Connecticut. U.S.A.. 1971. G. Roncari, H. Zubcr and A. Wyttenbach. 111r. J. Peptitle Prorriu RL’s.. 4 (1972) 267. A. M. Bond and D. R. Canterford, Aurrl. Clrern.. 44 (1972) 721. K. E. Zachu, M. P. Bmtzcl. Jr., J. D. Wincfordner and J. M. Mnnsficld. Jr.. Alla/. Clrcw.. 40 (1968) 1733. H. A. Schrocdcr and A. P. Neson. C/h. C/IO~L. I7 (1971) 461.