Determination of picogram quantities of zinc in zinc metalloproteins by atomic absorption spectrometry using a graphite furnace atomizer

ANALYTICAL

BIOCHEMISTRY

126,2 14-22 1 (I 982)

Determination of Picogram Quantities of Zinc in Zinc Metalloproteins by Atomic Absorption Spectrometry Using a Graphite Furnace Atomizer TARAHIRO

KUMAMARU,’

JAMES

F. RIORDAN,

AND BERT

L. VALLEE~

Center for Biochemical and Biophysical Sciences and Medicine, and the Department of Biological Chemistry, Harvard Medical School, and Brigham and Women’s Hospital, Boston, Massachusetts 02115 Received May 12, 1982 Optimum operating conditions have been determined for the atomization of zinc from metalloproteins in a graphite furnace. Addition of 50 mM ammonium dihydrogen phosphate to the protein and measurement of the integrated absorbance suppresses or eliminates matrix interference effects. Using a 5-~1 sample both the sensitivity and the detection limit are 0.3 ng of Zn/ml, i.e., 1.5 pg of zinc on an absolute basis. For 10 r&ml of zinc in 5-~1 samples of a zinc metalloenzyme, the coefficient of variation is 1.5%. Accuracy has been established by analysis of zinc metalloenzymes of known zinc stoichiometry. The method has been applied successfully to the determination of zinc in several proteins for which zinc stoichiometry had been unknown.

Zinc-containing enzymes are present in species from all phyla. They participate in a wide variety of metabolic processes including synthesis or degradation of carbohydrates, lipids, proteins, and nucleic acids (1,2). Any investigation of the biochemical function of zinc including its role in such zinc metalloenzymes requires highly precise, rapid, and convenient means for analyzing the metal in a complex, organic matrix. Present methods such as flame atomic absorption spectrometry are generally adequate when milligram amounts of enzyme are available. However, in the case of many enzymes, only microgram amounts can be obtained. Microwaveinduced plasma emission spectrometry has been employed under such circumstances, (3) and an improved method is reported in the previous paper (4). The technique requires the removal of significant amounts of salts and extraneous reagents from the protein sample to eliminate matrix effects ob’ Present address: Hiroshima University, Faculty of Integrated Arts and Sciences, Hiroshima 730, Japan. * To whom correspondence should be addressed: Seeley G. Mudd Bldg., 250 Longwood Ave., Boston, Mass. 02115. 0003-2697/82/l

502 14-08$02.00/O

Copyright 0 1982 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

214

served during metal determination. In recent years graphite furnace atomic absorption spectrometry has gained popularity due to its high sensitivity and the simplicity of operating procedures. Several studies have used this technique to determine zinc values in such biological samples as parotid saliva (5), cerebrospinal fluid (6), and silkworm eggs (7) but investigations of zinc metalloenzymes homogeneous by physicochemical criteria and of known stoichiometry are still limited. Moreover, means have not been found to eliminate the pretreatment of biological samples in order to remove matrix interferences. We here draw attention to matrix interferences which are encountered often in routine analyses of zinc metalloenzymes and show how they can be eliminated or sup pressed. EXPERIMENTAL

Apparatus. A Perkin-Elmer Model 5000 atomic absorption spectrophotometer equipped with a Perkin-Elmer Model HGA500 graphite furnace and a Model 056 strip chart recorder was used. Pyrolytically coated

ZINC DETERMINATION

BY ATOMIC TABLE

ABSORPTION

215

SPECTROMETRY

1

ATOMICABSORPTIONPARAMETERS Spectrophotometer operating conditions (Perkin-Elmer Model 5000) PE Zinc Intensitron lamp, 213.9 nm, 15 mA, slit L 0.7 nm Deuterium compensation, AA-BG mode Absorbance measurement, peak height and peak area modes Integration time, 6 s (atomizing step) Graphite furnace program (Perkin-Elmer Model HGA-500)

Temperature, “C Ramp time, s Hold time, s Internal gas flow, ml/min

Step 1 (Dry@9

Step 2 (Ashing)

120 5 25 300

500 5 60 300

Step 3 (Atomizing) 2400 2 4 50

Step 4 (Conditioning) 2600 1 3 300

Sample volume, 5 ~1

graphite tubes (Perkin-Elmer Corp.) were used in the furnace. Background emission correction was accomplished by using a deuterium lamp. In all cases, 5-pi aliquots were introduced into the furnace using a micropipet (Oxford Laboratories, Ultramicro Sampler Pipetting System) fitted with a disposable plastic tip. This type of pipet improved precision by preventing contamination and/or sample loss owing to the tip of the pipet touching the inside wall of the graphite tube injection port. Both peak height and integrated absorbance were measured, and the results of each were compared. All laboratory glass and plastic ware was cleaned thoroughly with 4 M nitric acid and checked for zinc content prior to use. Reagents. Unless stated otherwise, all chemicals used were of analytical reagent grade. Dilutions were made with demineralized water from a Pyrex still (Barnstead Co., 4 liters/h) fed with tap water that had been passed through a string filter (Commercial Filter Co., BRX 8), a colloid-removing filter, and a mixed-bed, ion-exchange filter (Barnstead Co.). Zinc standards (pH 2) containing 1000 pg of Zn/ml, were prepared by dissolving 0.6224 g of Specpure grade zinc oxide (John-

son-Matthey Co.) in dilute nitric acid (Alfa Products) and adjusting to a volume of 500 ml. Ammonium dihydrogen phosphate solution was prepared by dissolving ammonium dihydrogen phosphate to a concentration of 1 M and purified by solvent extraction with 0.03% dithizone in carbon tetrachloride. Ultrapure grade nitric acid was mixed with water to give a concentration of 1 M. Argon of 99.996% purity (Union Carbide Co.) served as the purge gas for the graphite furnace. Procedure. A sample for analysis is mixed with 1 M nitric acid, 1 M ammonium dihydrogen phosphate solution, and an appropriate amount of water resulting in a solution containing 5-20 ng Zn/ml, 10 mM nitric acid, and 50 mM ammonium dihydrogen phosphate. Five microliters of the sample solution are transferred to the graphite tube for each analysis. The operating conditions for the atomic absorption spectrophotometer and the graphite furnace unit are shown in Table 1. All steps subsequent to the injection of the sample proceed automatically in sequence according to the program, and the peak height (peak absorbance) and the peak area (integrated absorbance) are displayed as digital outputs. All analyses are performed in duplicate to improve precision. The final

216

KUMAMARU,

RIORDAN,

AND VALLEE

I

4 ASHING

TEMPERATURE,‘C

FIG. I. Effect of ashing temperature on the peak (A) and the integrated area (B) absorbances of Zn. 50 mM NbH2P04- 10 mM HNOj solutions of 10 rig/ml (0.153 PM) Zn standard (0), 0.125 *M CPD-A (O), 3.79 X IO-’ jtM ADH (A), and 3.39 X lo-’ pM AP (A), 10 mM HNOS solution of 10 rig/ml Zn standard (0).

operating conditions were determined on the basis of the experimental results discussed below. RESULTS AND DISCUSSION

Optimization of Atomization Conditions Zinc salts and several zinc metalloenzymes in buffers, i.e., alkaline phosphatase (AP)3 in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), carboxypeptidase A (CPD-A) in sodium borate and sodium chloride, and alcohol dehydrogenase (ADH) in sodium dihydrogen phosphate, all served as standards of reference. Each solution contained approximately 10 ng of Zn/ml, 10 mM nitric acid, and, except for one of the zinc standard solutions, 50 mM ammonium dihydrogen phosphate which is effective in eliminating matrix effects (see below). Drying. Empirically, a setting of 120°C is sufficient for all sample solutions. A ramp time of 5 s avoids abrupt vaporization of the sample and is followed by a 25-s drying step. As/zing.The effects of ashing temperature on peak heights and peak area are shown in Figs. 1A and B, respectively. In the presence ’ Abbreviations used: AP, alkaline phosphatase; CPDA, carboxypeptidase A; ADH, alcohol dehydrogenase; Hepes, N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid; Mes, 2-(YV-morpholino)ethanesulfonic acid.

of added phosphate, the peak height and peak area of the zinc standard and all the enzyme solutions tested depend to about the same extent on ashing temperature over the range of 400-600°C. An excess amount of ammonium dihydrogen phosphate relative to zinc increases the vaporization temperature of the zinc species to some degree. In addition, at temperatures below 3OO”C, peak absorbances of some test solutions are higher (Fig. IA), but their reproducibility is much lower, in part due to incomplete ashing of the sample constituents. Based on the above results, an ashing temperature of 500°C was selected for routine analysis. Atomization. Comparison of solutions in the presence and absence of phosphate revealed both a broad range of optimal temperatures and enhanced sensitivity, but only when peak height was measured (Fig. 2A). This effect of phosphate is not apparent in peak area measurements (Fig. 2B). The integrated signal did decrease slightly as atomization temperature was increased progressively over the temperature range tested (Fig. 2B). This behavior is the result of larger diffusional and convective losses of zinc atoms in the optical path due to increasing rates of rise in furnace temperature resulting from higher temperature settings. Conversely, slow atomization caused by low temperature settings lowers peak height and

ZINC DETERMINATION

OU 2000

BY ATOMIC

2200

2400

ABSORPTION

0)/h 2000

2600

ATOMIZING

TEMPERATURE,

217

SPECTROMETRY

2400

2600

2000

Y

FIG. 2. Effect of atomizing temperature on the peak (A) and the integrated area (B) absorbances of Zn. See Fig. 1 for conditions.

increases the half-width of the zinc signal. Figure 2 also shows that samples containing 50 mM phosphate have the same atomization behavior, regardless of sample composition. In practice, an atomization temperature of 2400°C was chosen both for peak height and peak area measurements. Under these conditions, a reproducible, single peak was obtained using 2 and 4 s for ramp and hold times, respectively. Step atomization, i.e., O-s ramp time, was not employed since it resulted in multiple peaks. To obtain higher sensitivity the internal argon gas flow was

decreased from 300 to 50 ml/min atomization step.

during the

Efect of Ammonium Dihydrogen Phosphate Ammonium or sodium hydrogen phosphate enhances the peak height absorbance of zinc. Similar behavior was also observed on addition of potassium or ammonium sulfate, and even sulfonic acids such as N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic

TABLE 2

-NH.,HzPOd Added compound NaCl &SO4 WUSO4 NaNO, Na borate Na acetate Na citrate Hepes MC%

Tris Veronal

1 mM -15 0 t20 +20 0 0 0 0 0 -10 +35

10

mM

-55 +10 t30 t10 -65 0 0 +10 f25 -15 +30

t50 mM NI&H$Od

35mM

IrnM

-40 i-15 +35 t15 -55 -10 -15 t30 +15 -70 -25

0 0 0 0 0 0 0 0 0 0 0

“Zn*+: 10 ng/mI (0.01 M in HN09). b Expressed as 46 increase or decrease of zinc absorption signal.

10 rnM 0 0 0 0

-20 0 0 0 0 -35 0

35 mM -15 0 0 0 -35 -10 -20 0 -15 -40 -10

218

KUMAMARU,

RIORDAN,

AND VALLEE

w 0.2~z 2

-0.8 mo z 5 --0.4 g z E c

k 2 w” 5 01’ 0

I l, I 100 ” 300

50 [NH&P041,

mM

FIG. 3. Effect of concentration of NH4H2P04 on the peak and the integrated absorbances of Zn. Zn: 10 rig/ml; HN03: 10 mM; peak absorbance (0); integrated absorbance (0).

acid (Mes) gave similar effects. However, the peak heights are dependent to some degree on the amount of the sulfates or sulfonic acids added (Table 2). In contrast, over a concentration range of 5-300 InM, addition of ammonium dihydrogen phosphate enhanced the peak height by a factor of 1.3 and did not interfere with the measurement of the peak area (Fig. 3). The presence of phosphate in the sample solution would be expected to suppress potential interference by matrix constituents. Accordingly, metal ions and salts which occur in biological samples as well as various buffers were tested for their effects on zinc determination in the presence and absence of phosphate. For this experiment the phosphate concentration was kept at 50 InM for this experiment. Results obtained by both

methods of measurement are shown in Tables 2-4. The interactions are expressed as the percentage increase or decrease of the zinc absorption signal in the absence of interfering substances. Variations of less than 8% in the observed values are listed as zero. Sodium chloride, sodium borate, and Tris suppress the peak height absorbance of zinc, whereas ammonium sulfate, potassium sulfate, sodium nitrate, Hepes, and Mes enhance it. The addition of 50 mM ammonium dihydrogen phosphate effectively eliminates the interference of ammonium sulfate, potassium sulfate, sodium nitrate, Hepes, and sodium chloride (Table 2). Moreover, peak area integration was far more effective in reducing matrix interference, especially in the presence of added phosphate (Table 3). Compared to peak height, measurement of the

TABLE 3 THE EFFECX OF NH4H2P04 ON INTERFERENCEOF BUFFERS IN THE DETERMINATION OF ZINC BY THE PEAK AREA INTEGRATION METHOD"'~ -N&H2P0.,

+50 mM N&H2P04

Buffer

1 rnM

10 mM

35mM

1 nlM

10 mM

35 rnM

Na borate Tris

0 0

-60 0

-65 -60

0 0

0 0

0 0

a Expressed as % decrease of signal. b Independent of the presence of phosphate, the following do not interfere at concentrations up to 35 mM: NaCl, KzS04, (NI-I&S04, NaNOS, Na acetate, Na citrate, Hepes, Mes, Veronal; Zn*+: 10 &ml (0.01 M in HNO3).

ZINC DETERMINATION

BY ATOMIC

ABSORPTION

219

SPECTROMETRY

TABLE 4

-NH.,H$Q Ion

Added as

MI?

Mg (NW2

Ca2+ Mn2+ Fes+ co2+ Ni*+ cu2+ Cd’+ Hg2+ As (III) Se (IV) MO (VI)

Peak height

Peak area

Peak height

-30

0 -10 -15 +35 -10 0 0 0 0 0 +10 0

+10 0 +10 +15 0 0 0 0 0 0 0

0 +20 +40 +25 +10 +30 +20 0 +10 +15 +15

Qa2

Mn(NW2 I-% WNW2

NiWNz WNW2

WNOd2 I-&Cl2

H,AsOs H2w3

HzMo04

+50

mM

m2m4 Peak area

0

0 0 0 0 0 0 0 0 0 0 0 0

’ Zn2+: 10 r&ml; inorganic ions: 10 pg/ml; HNOs: 0.01 M. * Expressed as % increase or decrease of zinc absorption signal

peak area absorbance is much more effective in obviating interference by inorganic ions. If both area integration and phosphate addition to the sample solution are employed, effects of numerous ions can be removed completely, even when present in a lOOO-fold (by weight) excess over zinc (Table 4). As described above, addition of ammo-

nium dihydrogen phosphate increases the effective ashing temperature for zinc sample solutions. This suggests that Zn(II) reacts with ammonium dihydrogen phosphate to form Zn3(PO& [mp 900°C (8); bp not measured], a compound thermally more stable than other zinc species, such as Zn(NO& [mp 360°C (9); bp not measured], ZnC& [mp

TABLE 5 DETERMINATIONOFZINCINZINCMETALLOPROTEINSOFKNOWNSTOICHIOMETRY g-atom Zn/mol protein Present method

MES”s*

Flame AAS*

3.39 X lo-’ pM protein 1 X lo-* mM Tris-HCl

4.1

3.7

3.6

Bovine carboxypeptidase A

1.25 X 10-l pM protein 5 X 10e2 mM Na borate 1 mM NaCl

1.0

1.0

1.0

Horse liver alcohol dehydrogenase

3.79 X loe2 pM protein 1.5 X 10m2mM Na phosphate

3.8

4.2

3.9

Zinc metalloprotein E. coli

alkaline phosphatase

Constituents

’ Microwave emission spectrometry. * Ref. (3).

220

KUMAMARU,

RIORDAN,

283°C (8); bp 732°C (S)], and ZnSO, [decomposition at 600°C @)I, prior to atomization. Although further experimental work is required to confirm the underlying mechanism, ammonium dihydrogen phosphate appears to serve as an effective masking agent for numerous interferents. Working cuwe. Calibration curves were prepared for samples from 5 to 20 ng of Zn/ ml in solution of 50 IIIM ammonium dihydrogen phosphate and 10 mM nitric acid. A linear calibration curve was obtained by the area integration method, while that of the peak height method has a small decrease in slope. Both the sensitivity and the detection limit were 0.3 ng of Zn/ml, i.e., 1.5 pg of zinc. Sensitivity is defined as the zinc concentration or weight resulting in an integrated absorbance of 0.0044 (absorbance X second) and the detection limit as the zinc concentration or weight required to result in an integrated absorbance signal twice that of the standard deviation of the background noise level. Accuracy and repeatibility. We have established the accuracy of the present method,

AND

VALLEE

combining phosphate addition with peakarea integration by analyzing metalloenzymes of known zinc stoichiometry. In particular, we have measured three types of purified metalloenzymes, i.e., AP from Escherichia coli, CPD-A from bovine pancreas, and ADH from horse liver. The results are shown in Table 5. The data are in excellent agreement with those obtained by microwave-induced emission and flame atomic absorption spectrometric methods (3), the latter employing much greater amounts of enzyme. Repeatability of the present method was estimated from 10 replicate determinations of zinc in a 2.94 X lop2 pM solution of squirrel monkey ADH (1 I). The mean integrated absorbance was 0.110 with a standard deviation of 0.00 16, i.e., a coefficient of variation of 1.5%.

Application to the Analysis of Zinc Metalloenzymes of Unknown Zinc Stoichiometry The method was applied to the determination of zinc in CPD of Streptomycesgri-

TABLE

6

DETERMINATION OF ZINC IN ZINC METALLOPROTEINS OF UNKNOWN STOICHIOMETRY Zinc metalloprotein

Constituents

g-atom Zn/ mol protein

1.63 X 10-l gM protein 5 /.LMTris-HCl 5 /.iM CaCl*

1.1

carboxypeptidaSe= Wr 4130) Squirrel monkey liver alcohol dehydrogenase b

2.94 X lo-* pM protein 1 mrvt Tris-HCl

4.0c

4.65 X lo-* @M protein 0.1 mM Tris-HCl 1 mM KC1

3.9

1.21 X 10-l pM protein 2 mM Tris-HCl

1.1

S. griseus

(Mr

78,430)

Squirrel monkey liver alcohol dehydrogenase ’ (Mr

76,230)

Mouse nerve growth factor’ CM 116,ooo)

a Ref. (10). * Sensitive to pyrazole inhibition (11). cCoefficient of variation = 1.5% in 10 consecutive measurements. d Insensitive to pyrazole inhibition (I 1). ’ Ref. ( 12).

ZINC DETERMINATION

BY ATOMIC

seus, two molecular forms of ADH from squirrel monkey liver, and nerve growth factor of mouse salivary glands. These proteins were dialyzed vs metal-free buffers and then diluted with 50 mM ammonium dihydrogen phosphate- 10 tnM nitric acid for zinc analysis. The results are also shown in Table 6. Moreover, 10 months use of the method in our laboratory has demonstrated its stability and reliability for the analysis of zinc metalloproteins. CONCLUSION

An atomic absorption spectrometric method using a graphite furnace atomizer has been employed for the determination of picogram quantities of zinc and provides a widely applicable technique for the analysis of zinc metalloproteins. Numerous interferences observed in the graphite furnace atomization process can be overcome by the addition of ammonium dihydrogen phosphate to the sample solution and by using the peak area integration method for absorbance measurements. Using this procedure, 5-100 pg (0.1-10 pmol) of zinc can be determined in less than 2 min with as little as 5 ~1 of sample. In addition, this method is superior to conventional methods due to its high sensitivity, microliter sample requirements, freedom from interferences, and simple operational procedures.

ABSORPTION

SPECTROMETRY

221

ACKNOWLEDGMENTS This work was supported by NIH Grants GM 15003 and HL22387 from the Department of Health and Human Services.

REFERENCES 1. Vallee, B. L. (1977) in Biological Aspects of Inorganic Chemistry (Dolphin, D., ed.), pp. 37-60, Wiley, New York. 2. Auld, D. S. (1979) in Ultratrace Metal Analysis in Biological Sciences and Environment (Risby, T. H., ed.), pp. 112-133, Amer. Chem. Sot., Washington D. C. 3. Kawaguchi, H., and Vallee, B. L. (1975) Anal. Chem. 47, 1029. 4. Kumamaru, T., Riordan, J. F., and Vallee, B. L. (1982) Anal. Biochem. 126,208-213. 5. Henkin, R. I., Muller, C. W., and Wolf, R. 0. (1975) J. Lab. C/in. Med. 86, 175. 6. Mazzucotelli, A., Galli, M., Benassi, E., Loeb, C., Ottonello, G. A., and Tanganelli, P. (1978) Analyst 103, 863. 7. Fujiwara, K., Umezawa, Y., Numata, Y., Fuwa, K., and Fujiwara, S. (1979) Anal. Biochem. 94, 386. 8. Weast, R. C. (ed.) (1978) CRC Handbook of Chemistry and Physics, 59th ed., pp. B182-B183, CRC Press, Boca Raton, Fla. 9. Sidgwick, N. V. (1962) The Chemical Elements and Their Compounds, Vol. 1, p. 276, Oxford Univ. Press, London. 10. Breddam, K., Bazzone, T. J., Holmquist, B., and Vallee, B. L. (1979) Biochemistry 18, 1563. 11. Pares, X., Dafeldecker, W., Vallee, B. L., Bosron, W. F., and Li, T.-K. (1981) Biochernistry20,856. 12. Young, M., and Koroly, M. G., personal communication.