SPIN-LABELING TECHNIQUES FOR STUDYING 



MODE OF ACTION OF PETROLEUM 



HYDROCARBONS ON MARINE ORGANISMS 



William T. Roubal' and Tracy K. Collier^ 



ABSTRACT 



Spin-labeling studies of membrane-contaminant interaction are being conducted by biochemists at the 

 Northwest Fisheries Center in Seattle, Washington. The aim of these studies is to gain a better 

 understanding of the mode of action of hydrocarbon contaminants at the molecular level. Basic 

 spin-labeling theory together with experimental results are presented and discussed. Spin-labeling 

 holds great promise not only for environmental studies but also for drug research, toxicology, and 

 pharmacology as well. 



The interaction between contaminants and living 

 systems commences when contaminants combine 

 with so-called active sites in living tissue. Active 

 sites are varied in nature, but often they are 

 groups of molecules assembled in a special fashion 

 such as those which comprise membranes and as- 

 sociated enzymes or other biopolymers. 



Although the exact nature of contaminant-host 

 interaction may not be known in each and every 

 case, experimental data from biochemical /bio- 

 physical studies allow us to draw certain 

 conclusions about interactions. With detailed 

 investigations, collected data may even allow us to 

 draw a fairly accurate picture concerning the 

 molecular basis of physiological changes which 

 contaminants are able to induce. 



Admittedly, investigations such as these are 

 difficult to perform. The molecular complexity of 

 living systems defies ready characterization, and 

 it is even more difficult to relate alterations in 

 molecular organization to the subsequent 

 physiological changes wrought by this con- 

 taminant-host interaction. 



Recent years have seen an upsurge of interest in 

 membranes and how membrane structure is 

 modified when invaded by such things as drugs 

 and insecticides. The reasons are several: 

 membranes house cells, control the influx and 

 efl!iux of nutrients and metabolites; membranes 



'Northwest Fisheries Center, National Marine Fisheries Ser- 

 vice, NOAA, 2725 Montlake Boulevard East, Seattle, WA 98112. 



^Northwest Fisheries Center, National Marine Fisheries Ser- 

 vice, NOAA, 2725 Montlake Boulevard East, Seattle, WA 98112, 

 and The College of Fisheries, University of Washington, Seattle, 

 WA 98195. 



Manuscript accepted July 1974. 



FISHERY BULLETIN: VOL. 73, NO. 2, 1975. 



control and form the basis for the transmission of 

 electrical signals (nerve impulses) when nerve 

 receptor sites are stimulated. Any major altera- 

 tion to normal membrane structure may be ex- 

 pected to play some role in animal physiology, 

 especially if the contaminated membrane is 

 associated with neural function or other viable life 

 processes. 



Membranes consist of a sandwich of 

 phospholipids, sterols, and proteins; individual 

 membranes are microscopically thin. The thinness 

 and complexity of membranes make their study 

 most difficult. One way of characterizing 

 membranes is by measuring their electrical 

 properties (conductance, resistance, capacitance, 

 etc.). However, with respect to contaminant-host 

 interaction, it is more desirable to be able to 

 deduce structural features such as lipid fluidity, 

 protein-lipid interaction, and arrangements of 

 constituents in dynamic tissue preparations. This, 

 then, precludes the use of electron microscopy or 

 other methods which are incompatible with main- 

 taining tissue in a viable unchanged condition. 



THE THEME OF THIS REPORT- 

 SPIN-LABELING 



Several years ago, it was observed that certain 

 free radical derivatives of fatty acids could be in- 

 troduced into membrane preparations without 

 unduly disturbing the natural arrangement of 

 native membrane constituents (Jost et al. 1971). 

 Furthermore, it was shown that these free radicals 

 would associate with and align in membranes 



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