Damage to ATP by peroxidizing lipids

402

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BBA 23 328

Damage to ATP by peroxidizin 9 lipids Following the determination of protein and enzyme products in model systems containing peroxidizing lipids ~-3, similar studies were made with ATP as an example of a nucleotide. Similarities exist between peroxidation and ionizing radiation, a process fairly well characterized; both lipid peroxidation and ionizing radiation produce free radicals that cause biochemical damage. An emulsion of ethyl arachidonate and phosphate buffer containing ATP was peroxidized to the extent of 2.4 moles O,)/mole arachidonate by shaking the emulsion in O,, at 37 °. The reaction mixture consisted of o.22 g ethyl atachidonate and o.o5 g ATP in o.5 ml o.o5 M KH2PO4--Na2HPO 4 buffer (pH 7.o). Water-soluble pioducts of the reaction were chromatographed on thin layers of cellulose powder (MN 3oo(i, Macherey, Nagel and Co.) developed with tert.-amyl alcohol--formic a c i d - w a t e r (3 : 2 : i, v/v/v). Spots were detected by spraying the plates with o. 1% aqueous potassium permanganate, then exposing the plates for I5 sec to chlorine, and subsequently spraying them with 3 M potassium hydroxide. Automated Sephadex gel filtration 4,'~ was also employed for fractionation of the water-soluble products from separate peroxidation reactions of both [iI-~Cilinolenate and unlabelled ethyl arachidonate. Colunm effluent was monitored with a deep-path ultraviolet-transmitting flow cuvette 6 and an assembly of Photovolt components. Details of the methods as well as a description of components have been published elsewhere2,3, ~. On the basis of peak areas, the amount of ATP destroyed per mole of peroxide formed was determined using the relationship: ATP destroyed ~ - wt. AFP % nonNl'P/mol. wt. ATP ~'4 moles peroxide formed, assuming that the extinction coefficient of non-ATP products did not differ markedly from that of ATP itself. The thin-layer chronlatographic studies indicate the presence of adenosine, AMP, and ADP (Table I). Separations are expressed as relative mobility with respect to adenosine (Ra). TABI,E TItIN

1

LAYER

CHROMATOGRAPHY

f)l ~ \V/kTI~;R-SOLUBLE

:\T})--IUFHYL

ARACHII)ONA'FF~

RIL:kCTION

PRODUCTS

Standards

Adenosine AMP ADP ATP

Color after spray

17,

Pink Pink Pink Pink

i.o o.() 5 o.32 o. I O

Neacgion mixture l?c~

Color

i. 1 o.O 3 o.30 o. 17

I'ink Pink Pink Pink

Sephadex gel filtration confirmed the presence of AMP and ADP together with :a species having a molecular weight of 7oo. A simple free-radical addition polymerization, as in the case of protein-lipid peroxidation products, does not appear to be involved, since compounds of tool. wt. 7oo are not whole nmltiples of ATP. The soluble ATP reaction product, isolated via Sephadex gel filtration, conBiochim. Biophys. Acta, 136 (1967) 402 403

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tained a low level of incorporated lipid; approx. 4" IO-* mole linolenate was incorporated per mole of equivalent ATP. Although the extent of oxidation reached a limiting value with linolenate, lesser amounts of ADP and AMP were formed with linolenate than with arachidonate. The low amount of lipid incorporated and the fact that both chromatographic techniques substantiate the formation of ADP and AMP indicate that cleavage of phosphate is a predominant reaction in peroxidizing l i p i d - A T P mixtures. ADP reaction products were investigated by the use of Sephadex gel filtration only. The only difference between the reaction products and the control was the existence of a small peak in the molecular weight region of ATP. It is evident that peroxidizing lipid-nucleotide interactions lead to products of both increased as well as decreased molecular weight. In contrast to peroxidizing lipid-protein studies, however, the nature of the polymeric material and the pathway leading to such products have yet to be elucidated. In a study of ADP, HEMS has shown that 0.3 mole of this material is destroyed per ion pair ~, or about IO times the damage noted in the case of equivalent lipid peroxide-protein interaction. The damaging efficiency of lipid peroxidation for nucleotides, on the other hand, was much less--namely, 2. IO 5 mole ATP destroyed per mole of free radicals. Qualitatively, the picture for ionizing radiation and peroxidation damage is similar; less ADP and AMP have been noted in the case of irradiated ATP where most of the product is adenine.

Food Science Pioneer Research Laboratory, U.S. Bureau of Commercial Fisheries, Seattle, Wash., and Department of Food Science and Technology, University of California, Davis, Calif. (U.S.A.) I 2 3 4 5 o 7

W. T. ROUBAL

A. L. TAPPEL

1. D. DES.aI AND A. L. TAPPEL, J. Lipid Res., 4 ( i 9 6 3 ) 204. \V. T. ROUBAL AND A. L. TAPPEL, Arch. Biochem. Biophys., l I 3 (1966) 5\V. T. [)vOUBAL AND A. L. TAPPEL, Arch. Biochem. Biophys., I t 3 ( I 9 6 6 ) I5O. \V. T. ROUBAL AND A. L. TAPPEL, Anal. Biochem., 9 (1964) 211. \V. T. ROUBAL, P h . D . Thesis, U n i v e r s i t y of C a l i f o r n i a , D a v i s , Calif., 1 9 6 5 . W . T. ROUBAL, Anal. Chem., 37 (1965) 44 °. (~'" HEMS AND ~l. L. EIDINOFF, Radialion Res., 9 (1958) 305 •

Received October 3Ist,

I966 Biochim. Biophys. Acta, 1 3 6 (1967) 4 0 2 - 4 0 3