Iron in commercial ATP preparations

SHORT

525

COMMUNICATIONS

Iron in commercial ATP preparations Commercial preparations of ATP have been vastly improved in respect to purity since their initial entrance onto the market. As a result, investigators are often confident enough to use them in metabolic or enzymic studies without further purification. Based on the fact that the major impurities are breakdown products such as ADP and Pi, their use would not seem to entail a great risk since these contaminants can be accounted for in the usual assay procedures. However, there is always the possibility of the presence of uncharacterized, biologically active contaminants in impure preparations which could interfere with the biochemical system under study. A discovery of LORAXD et a1.l serves as a good example of this; they found variability in susceptibilities to hydrolysis of commercial ATP by myofibrils and actomyosin and traced it to calcium which they showed to be a contaminant in some commercial ATP preparations.

Another

example

was provided

by HOCHSTEIN

et aL2 who found

small and variable but detectable quantities of iron in commercial ATP and quantities of iron in some ADP preparations sufficient to have a considerable effect on ADPactivated peroxidation of lipids in microsomes and mitochondria. In a current investigation of the interaction of commercial ATP with catecholamines we have encountered 10-20 times more iron contamination than reported by HOCHSTEIN et aL2. In this note we describe the initial observation together with the identification of the metal and a survey of the iron content of commercial ATP using calorimetric and atomic absorption assays. The observation which led to the finding of a metal ion contaminant was the detection

of blue and pink color formation

upon interaction

of equimolar

amounts

of noradrenalin with various commercial ATP preparations. The colors were visible to the eye when the noradrenalin and ATP concentrations were >O.OI M and when the pH was >5.5. A pH study showed reversible transitions from colorless to blue in the pH range 5.5-6.5, and from blue to pink in the pH range 6.5-8.0, similar to the reversible color changes typical of pH indicator dyes. At pH 7.3, the pink solutions absorbed maximally at 500 nm and, at pH 6.5, the blue solutions absorbed maximally, with a broad peak, at 570-610 nm. Other catecholic compounds tested included adrenalin, dopamine, and pyrocatechol; each induced the color formation but the pH profile in respect to the appearance of blue and pink color differed slightly in each case. Although, the absorption spectra of the noradrenalin oxidation product, noradrenochrome, and the pink product observed in this study were found quite similar, their identity was ruled out on the basis of an observed lack of sensitivity of the noradrenochrome spectrum to pH in the pH range 5.0-7.0. The color response of noradrenalin with each ATP preparation was found linear in respect to the concentration of each ATP sample and there was a wide range of response (Table I). A mYtalion contamination capable of interaction with the catecholamine was suspected since colored complexes of catecholic compounds and metal ions are known3$4. EDTA was found to cause complete disappearance of the color and this observation partially confirmed the suspicion of metal ion contamination. Systematic trials with various metal ions indicated that the color-inducing factor is iron since mixtures of catecholic compounds with Fez+ or Fe3+ resulted in products similar to the products which were formed upon interaction of the catechols with the ATP Biochim. Biophys. Acta, 192 (1969)

525-527

526

SHORT COMMUNICATIONS

samples; identity was based on spectral characteristics and pH profiles of color transitions. To identify the metal in a more standard way as well as to quantitate it and determine its valence state, the reagent, bathophenanthrolinedisulfonic acid, which forms a pink-colored complex specifically with Fe2+ was employed5. When mixed with ATP this reagent did not induce color formation but when a reducing agent, hydroxylamine, was used in conjunction with it, the color developed, indicating that Fe3+, not Fe2+, was the contaminant5. Presumably, the catechols have dual roles in their interaction with Fe3+; i.e. they reduce it and they also complex with the reduced TABLE COLOR

I OF

EQUIMOLAR

MIXTURES

OF

ATP

AND

NORADRENALIN

The absorbances at 500 nm of solutions containing 0.033 M noradrenalin bitartrate and 0.033 M ATP in 0.166 M sodium phosphate buffer (pH 7.0) were measured with a Bausch and Lomb Spectronic 20 calorimeter. Available identification numbers of the ATP samples are given together with names of the suppliers. A TP (d&odium salt)

A500

Nutritional Biochemical Corp., 7554 Pabst (P.L.), 187-A Nutritional Biochemical Corp., I IOI Calbiochem, 71 105 Sigma, * *8B-7350 Pabst (P.L.), 34216-143-A Schwarz, 6804-D Sigma, 48B-1570

0.62 0.66 0.39 0.43 0.13 0.07 0.05 0.03

TABLE

nm

II

IRON CONTENT OF COMMERCIALATP PREPARATIONS The calorimetric determination of Fe a+ in ATP involved measurement with a Bausch and Lomb Spectronic 20 calorimeter of the absorbance at 535 nm of mixtures composed of: 12.0 mM ATP, 0.24 M sodium acetate buffer (pH 6.5), 0.36 mM disodium bathophenanthrolinedisulfonate, 0.034 mM hydroxylamine hydrochloride. The Fe3+ was estimated from a standard curve obtained by using FeCl, as a standard in place of ATP. Available identification numbers of the ATP samples are given together with names of the suppliers. Atomic absorption assays were run on the five samples indicated. Figures in parentheses are ppm. F&+ (mmoles/mole

d TP (disodium salt)

Calorimetric Nutritional Biochemical Corp., 7554 Pabst (P.L.), 187-A Nutritional Biochemical Corp., I IOI Calbiochem, 7r 105 Mann, 52 873 Sigma, r 18B-7350 Pabst (P.L.), 34216-143-A Schwarz, 6804P Sigma, 48B-1570 Nutritional Biochemical Corp., 7g87*

l

Dipotassium

salt.

Biochim. Biophys. Acta, 192 (1969)

525-527

4.8 5.2 2.8 3.3 2.5 0.3 0.4 0.2 0.2 0.1

A TP)

Atomic absorption

4.7 (432) 3.1 (291) 3.5 (317) 0.6 (57.4) 0.4 (40.6)

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527

product, Fe 2+, to form the colored products. To verify the results of the calorimetric assay with bathophenanthrolinedisulfonic acid, samples were selected for atomic absorption analysis by a commercial laboratory (Stewart Laboratories, Incorporated, Knoxville, Tenn.). A summary of the results of the calorimetric and atomic absorption assays is given in Table II. In addition to the results described in Table II, the atomic absorption studies also indicated that the iron contamination was not associated with other metal ion contamination, such as copper, as is often the case for a general contamination. The presence of Fe3+ in numerous commercial samples of ATP presents a problem to the biochemist. The misleading colors found on mixing ATP and catecholamines attest to this. The contaminating as a catalyst in electron transfer reactions,

iron could function, at the levels found, in oxygen binding, activation or reduction

processes as well as in a host of other reactions. Iron could interfere with numerous analytical procedures involving ATP such as NMR studies; it is possibly the unidentified interfering metal ion contaminant which STERNLICHT et aL6 removed in a Chelex-Ioo ATP purification procedure of metal-ion binding to ATP.

they found necessary

prior to a NMR stud]

The fact that other metals were not found associated with iron suggests that it may be bound to the ATP in some specific manner; stable complexes of ATP and Fe3+ have been demonstrated by GOUCHER AND TAYLOR’. The question also arises whether the Fe3+ is derived from the biological material from which the ATP is isolated. A recent report indicated that 20-40~/~ of the ATP of hemolysates and whole blood is complexed with irons. Previous investigations involving ATP should be reassessed in light of finding and investigators should be alert to the possibility of this contamination remove it if found. The manufacturers should take immediate steps to remove from their preparations if it is present in above background levels. It should be

this and iron em-

phasized that the study involved currently available preparations; the survey was not intended to be complete nor to reflect purity of past preparations or of current preparations not analyzed in this study. This work was supported in part by a grant from the Chicago and Illinois Heart Association. De@vtments of Neurology and Biochemistry, Presbyterian-St. Luke’s Hospital and Uniuevsity of Illinois College of Medicine, Claicr~go, Ill. 60 612 (U.S.A.)

W. H. HARRISOS R. M. GRAY T. DECLOUX

I L. LORAND, R. DEMOVSKY, J. MEISLER AND J. MOLNAR, Biochim.Biophys.Acta, 77 (1963) 679, 2 P. HOCHSTEIN, K.NORDENBRAND AND L. ERNSTER, Biochem. Biophys. Res.Comrnun., 14 (1964) 323. 3 C. XSGEL, B. CARPENTER, C. R. LAFFERTY AND B. S. BURKETT, Diseases Nervous System, LI (1960) 440. 4 J. W. MAAS AND R. IV. COLBURN, Nature, 208 (1965) 41. =, D. BLAIR AND H. DIEHL, Talanta, 7 (1961) 162,. g H. STERNLICHT, R. G. SHULMAN AND‘E. V?. ANDERSON, J. Chem. Plays., 43 (1965) 3123. 7 C. K. GOUCHER AND J. F. TAYLOR, 1. Biol. Chem., 239 (1964) 2251. b K. KONOPKA, W. LEGKO, R. GOND~O, 2. SIDORCZYK~ ~.FABJANOWSKA AND M. SWEDOWSKA, Clin.Chim. Ada, 24 (1969) 359. Received

September

Igth,

1569 Biochim. Biophys. Acta, 192 (1969) 525-527