ROUBAL and COLLIER: SPIN-LABELING TECHNIQUES 



with polar headgroups and nonpolar tails of car- 

 bon-hydrogen chains. Thermodynamically, and for 

 other reasons as well, the greatest stability for 

 membrane structure results when membrane con- 

 stituents are arranged as shown; polar surfaces 

 are exposed to aqueous environments, and the 

 fatty acid tails are tucked away out of contact with 

 water. 



QUESTIONS ABOUT 

 CONTAMINANT-HOST INTERACTION 



We would like to answer the following questions 

 concerning membrane-contaminant interaction: 

 a) are certain regions of membranes affected or is 

 the whole membrane affected when invaded by 

 contaminant? b) if the effect is localized, where is 

 the localization? c) are there differences in 

 membrane perturbations when treated on the one 

 hand by paraffins, and aromatics on the other? d) 

 can the differences, if they exist, be related to 

 anything presently known about the toxicology of 

 any of these contaminants? 



EXPERIMENTAL METHODS 

 AND RESULTS 



The study was performed in two steps. First, an 

 in vivo feeding study was undertaken. Here we 

 used spin-labeled hydrocarbons (Roubal 1974) and 

 fed them to fish. Second, an in vitro study was 

 employed using excised tissue. In order to restrict 

 our investigations of membranes (in vitro) to 

 specified regions, the series of labels I, II, and III 

 (Roubal 1974a) (Figure 6) were synthesized by the 

 EPR group of this Center. The positively .charged 

 quartemary nitrogen of label I directs this portion 

 of the label to the polar membrane surface, which 

 in turn insures that the nitroxide nitrogen is sit- 

 uated at or very near to the membrane surface. 

 The carboxyl groups of labels II and III direct the 

 carboxyl end of these amphiphiles to the 

 membrane surface, but now the nitroxide nitrogen 

 Ues some 12-15 A below the membrane surface in 

 II, and deep into the hydrophobic membrane 

 interior in III. Thus we can "look" at the 

 membrane's surface, subsurface, and interior. 



In the feeding study, spin-labeled hydrocarbons 

 were incorporated into fish food and fed to coho 

 salmon, Oncorhynchus kisutch, fingerlings in a 2- 

 day feeding study. Within an hour, or even less, 

 after onset of intake of food by fish, the blood 

 showed EPR activity. Using radiotracers, we have 



shown this activity to be associated with blood 

 lipoproteins and albumins (Roubal 1974b), with 

 lipoproteins making the greatest contribution to 

 hydrocarbon transport in blood. 



After an induction period of about a day, blood- 

 associated labels (Roubal 1974b) slowly transfer to 

 neural tissue and flesh. Weight for weight, the 

 greatest concentrations are to be found in the 

 spinal cord, lateral line nerve bundles, and brain. 

 The nature of the EPR line shapes indicated that 

 the invasion of hydrocarbon is site selective. All 

 labeled paraffins appeared to intercalate with 

 membrane in such a way that the nitroxide 

 mobility is little impeded. In direct contrast to 

 this, mobility of aromatics appeared to decrease. 

 This in vivo study suggests that paraffins associate 

 with molecularly fluid portions of membrane fatty 

 acids, while aromatics associate with the more 

 structured and rigid regions of membranes. In 

 order to clarify these possible differences, an in 

 vitro study was undertaken using labels I, II, and 

 III (Roubal 1974a). 



Neural tissue from untreated fish was carefully 

 excised and placed in cold, 0.1 M phosphate buffer, 

 pH 7.4 at 4°C, isotonic in NaCl. A sonicated dis- 

 persion of label, complexed to bovine serum al- 

 bumin was added and allowed to transfer to neural 

 membrane (overnight at 4°C). Membranes were 

 then inserted directly into the EPR spectrometer, 

 and the spectra were recorded immediately. After 



ar 



CH^CH^IjiN 





N, N- Dimethyl- 4- dodecyl-4-tempoyl-annmonium bromide 



n. 



m. 



0^ ^/v-o 



CH^-fCH^IjgC - (CH2)g COOH 



"7- Nitroxide" stearic acid 



ri: 



O ^N-O 



"12- Nitroxide" stearic acid 



Figure 6.-Lipid-intercalating spin-labels. 

 Label I reports on surface conditions. 

 Label II reports on subsurface conditions. 

 Label III reports on interior conditions. 



303 



