YHORT COMMUNICATIONS 367

Determination of Traces of Calcium in Commercial Adenosine Triphosphate (ATP)l

In our studies of the effects of cat’ions on the “ATP’:M~” activity of erythrocyte ghosts (1) it is important to UC preparations of ATP that are substantially free of Ca and Mg. Lorand et al. (2) reported finding 0.0009-0.02% Ca, by spect,ral analysis, in various commcrcinl prcpnra- tions. Seidel and Gergely (3) found about 0.2 mole s, by EDTA titrn- tion with calcein.

We found that the fluorometric determination of C:t in ATY with calcein, and direct spectrophotometric determination with various intli- caters for Ca, could not bc used, because ATP competes \vith the indi- cat,ors for Ca++ in solution.

We finally adopt’ed a modification of the differential qwctrophoto- metric method of Radin and Granza (4) with eriochrome blue SE’ as indicator. Cn is titrated in t’he spectrophotometer cuvet. with standard EGTA solution.” Schmid and Reiiley (5) had shown that the Cat-- EGTA complex has a wry high stability co&ant (log I< = 10.7~. Seiclel and Gergely (6) titrated the Cn in 0.04-0.08 X ATP solutions with EGT-1 and calcein visually, and could detect 10 ~11 Ca in this way.

The indicator solution, O.lOM in 0.5OM KOH (4), 3.00 ml, was placed in each of two cuvets (1.00 cm light path) and absorbnncee (.I1 were rent] at 615 n1/1. with the Gilford model 220 absorbance indicator.” A calciulrl stnntlnrc1 curve (O-O.15 +lole CR in 3 ml) was linear rind almost itlcntiwl n-itI1 tll:it of Radin and Granzn. Since the abso~~t~ancc~ intiicator gins linear readings up to B = 3.01 we could read the unknown cur-& against either a water or an indicator blank, and tlir differcncc$ were proportiom~l to Cn concentration.

Dil;;odium ATP ( lo-15 ,~nnolcs) was weighed on a microbalance and dissolvccl tlircctly in 3.00 ml of the indicator solution. Standard 10.0 m31 EGTA ww then acltlc(l from n syringe microburet to the ATP solu- t.ion and to the indicator blank. Absorbanccs against, water WCI~~ plot,tecI

against microiitcrs of EGTA solution. Titration curves were linear to the

‘Supported lay tlltl Medkal Research Council of Canada. ‘Mordant blue B, (2.1. 16680, So. 752 of the National Aniline Division of the

Allied Chemical Corp., I\;ew York, K. T. ’ [Ethylenebis(oxyethylenenitrilo) Itetrawrtic acid, No. 8276 of Distillation Prod-

ucts Industries, Rochester, N. T. ’ Gilford Instrument. I,:~bor:ltories, Oberlin, Ohio.

368 SHORT COMMUNICATIONS

equivalence point,, then became horizontal, with a sharp break at the end-point. The limit of sensitivity was about 0.01 pmole of Ca.

The results of titrations of several commercial products are given in Table 1. It is of interest that Sigma ATP, lot 22B-721, is the same lot’

TABLE 1 DETERMINATION OF CALCIUM IS COMYERCIAL ADESOSIXE TRIPHOWHATE

L-24470 0.40 “ZB-i21b 0.00 84B-7330” 0.87

a Mann Research Laboratories, Inc., New York, N. Y. b Sigma Chemical Company, St. Louis, MO. c Sigma, low calcium content. This preparat,ion did not give a sharp end-point and

may have contained other impurities.

that was used by Lorand et al. (2) in several kinetic measurements. They reported O.OOlc/c. Ca (0.15 mole %) in this lot.

Passage of our ATP solutions through small columns of Cheles 100, Na forn-i,s to remove divalent cations (cf. refs. 2, 3) decreased the Ca content to zero. Titration of one lot of monosodium adenosinc diphos- phatc (Sigma) revealed no calcium.

REFERENCES

1. MASN, J., AND COLLIEH, H. B., unpublished obscr\-ations. 2. LO~USI), I,., DRJIOVSKI-, Ii. d., MEISLI~H. J., AZD b~OLN.AR, J., Biochirn. Biophys.

Actn ‘77, 679 (1963). 3. SEIDEL, J., .zsn GERGELT, J., J. Bid. Chum. 238, 3648 (1963). 4. RADIS, H., AXI) C:R.4SZ:\, d. I,., (‘&,I. Chrt/t. 10, 704 (1964). 5. SCH~MID, R. W., AA-D REILIXY. C. S., Aural. L’hem. ‘29, 364 (1957). 6. SEIDEL, J., .ISD GERGEL~-, J., J. Bid. Chum. 239, 3331 (1964).

H.B. COLLIER G. DUCHON

’ Bio-Rad Laboratories, distributed by Calbiochem, I,os Angplps, Calif.