EPR MEASUREMENTS SHOWING THAT PLASMA-MEMBRANE VISCOSITY CAN VARY FROM30 TO 100 CP IN HUMAN EPIDERMAL-CELL STRAINS

A rigorous technique for the measurement of human membrane viscosity b y electron paramagnetic resonance (EPR) spectroscopy has been develope d by designing a sample preparation procedure to optimize the spin lab eling process and using a special (grown in essential fatty acid free medium) epidermal cell strain. The essential fatty acid deficient cell strains (keratinocytes) were also grown in fatty acid supplemented me dia formulated to alter the fatty acid composition of the phospholipid s that form the cell membrane. Fatty acid free bovine serum albumin wa s used as a carrier for the spin label (16-doxyl stearate methyl ester ) at an approximately equimolar ratio. Monolayers grown in T-75 flasks were labeled for 15 min at 4 degrees C with 12 mu M bovine serum albu min plus 20 mu M spin label. The cells were then washed and transferre d (at 4 degrees C) to a flatcell for EPR studies at 37 degrees C. The spectra were computer simulated and the results were interpreted by co mparison with a ''standard curve'' obtained from the EPR spectra of th e spin label in oil at multiple temperatures. Arguments are presented for preferring this measurement technique over the more conventional u se of order parameters and over the use of some other spin labels. The EPR spectra were completely insensitive to the effects of molecular d ioxygen in the growth medium and cytoplasm, but remarkabley sensitive to the fatty acid composition of the cellular phospholipids. Fatty aci d modified epidermal cells showed a very strong correlation between me mbrane fluidity (a three-fold change in the membrane viscosity) and a fatty acid double bond index.