EPR measurements showing that plasma membrane viscosity can vary from 30 to 100 cP in human epidermal cell strains

A rigorous technique for the measurement of human membrane viscosity by electron paramagnetic resonance (EPR) spectroscopy has been developed by designing a sample preparation procedure to optimize the spin labeling 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 media formulated to alter the fatty acid composition of the phospholipids that form the cell membrane. Fatty acid free bovine serum albumin was 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 albumin plus 20 mu M spin label. The cells were then washed and transferred (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 comparison with a ''standard curve'' obtained from the EPR spectra of the spin label in oil at multiple temperatures. Arguments are presented for preferring this measurement technique over the more conventional use of order parameters and over the use of some other spin labels. The EPR spectra were completely insensitive to the effects of molecular dioxygen in the growth medium and cytoplasm, but remarkabley sensitive to the fatty acid composition of the cellular phospholipids. Fatty acid modified epidermal cells showed a very strong correlation between membrane fluidity (a three-fold change in the membrane viscosity) and a fatty acid double bond index.