The membranes were centrifuged briefly, and the loose pellet was used for EPR measurements. The sample was placed in a 0.6 mm i.d. capillary made of gas-permeable methylpentene polymer, called TPX (Hyde and Subczynski 1989 ). Samples were thoroughly deoxygenated, yielding correct EPR line shapes and values of the spin-lattice relaxation time.
Conventional EPR spectra were obtained at 40°C with a Bruker EMX spectrometer with temperature control accessories. A temperature of 40°C was chosen to ensure that measurements were done above the phase transition temperature of ESM membranes (de Almeida et al. 2003 (link); Quinn and Wolf 2009 (link); Wisniewska and Subczynski 2008 (link)). EPR spectra were recorded with a modulation amplitude of 1.0 G and an incident microwave power of 5.0 mW. values were measured directly from the EPR spectra as indicated inFig. 1 . The order parameter was calculated as described in detail in Ref. (Marsh 1981 (link)). Because of the sharpness of the EPR lines and the method of measurements, values could be measured with an accuracy of ±0.1G, and the order parameter could be evaluated with an accuracy of ±0.015. Also, maximum splitting values could be measured with an accuracy of ±0.1 G, and the mobility parameter h+/h0 values with an accuracy of ±5%. To measure hydrophobicity, the z-component of the hyperfine interaction tensor of the n-PC or 9-SASL, AZ, was determined from the EPR spectra for samples frozen at −165°C and recorded with a modulation amplitude of 2.0 G and an incident microwave power of 2.0 mW (Subczynski et al. 1994 (link)). 2AZ values were measured within an accuracy of ±0.25 G.
The T1s of the spin labels were determined by analyzing the saturation-recovery (SR) signal of the central line obtained by short-pulse SR EPR at X-band (Subczynski et al. 1989 (link); Yin and Subczynski 1996 (link)) and used to draw fluidity profiles across membranes. The SR spectrometer used in these studies was described previously (Yin and Subczynski 1996 (link)). A relatively low level of observing power (8 µW, with a loop-gap resonator delivering an H1 field of 3.6×10−5 gauss) was used for all experiments to avoid microwave power saturation (which induces artificial shortening of the apparent T1). Accumulations of the decay signals were carried out with 2048 data points on each decay. SR signals were fitted by single- or double-exponential functions. When a single-exponential fit was satisfactory, the uncertainties in the measurements of decay time from the fits were usually less than 0.05%, whereas the decay times determined from sample to sample (for samples prepared totally independently) were within an accuracy of ±3%. When a double-exponential fit was necessary, and satisfactory, the decay times were usually evaluated with standard deviations less than ±5% and ±10% for longer and shorter recovery time constants, respectively. Larger standard deviations for shorter components are due to the difficulty in measuring very short T1s (due to the presence of molecular oxygen) in the current setting of the instrument. It is also possible that the available pump power cannot saturate the signal when the T1 is very short.
Conventional EPR spectra were obtained at 40°C with a Bruker EMX spectrometer with temperature control accessories. A temperature of 40°C was chosen to ensure that measurements were done above the phase transition temperature of ESM membranes (de Almeida et al. 2003 (link); Quinn and Wolf 2009 (link); Wisniewska and Subczynski 2008 (link)). EPR spectra were recorded with a modulation amplitude of 1.0 G and an incident microwave power of 5.0 mW. values were measured directly from the EPR spectra as indicated in
The T1s of the spin labels were determined by analyzing the saturation-recovery (SR) signal of the central line obtained by short-pulse SR EPR at X-band (Subczynski et al. 1989 (link); Yin and Subczynski 1996 (link)) and used to draw fluidity profiles across membranes. The SR spectrometer used in these studies was described previously (Yin and Subczynski 1996 (link)). A relatively low level of observing power (8 µW, with a loop-gap resonator delivering an H1 field of 3.6×10−5 gauss) was used for all experiments to avoid microwave power saturation (which induces artificial shortening of the apparent T1). Accumulations of the decay signals were carried out with 2048 data points on each decay. SR signals were fitted by single- or double-exponential functions. When a single-exponential fit was satisfactory, the uncertainties in the measurements of decay time from the fits were usually less than 0.05%, whereas the decay times determined from sample to sample (for samples prepared totally independently) were within an accuracy of ±3%. When a double-exponential fit was necessary, and satisfactory, the decay times were usually evaluated with standard deviations less than ±5% and ±10% for longer and shorter recovery time constants, respectively. Larger standard deviations for shorter components are due to the difficulty in measuring very short T1s (due to the presence of molecular oxygen) in the current setting of the instrument. It is also possible that the available pump power cannot saturate the signal when the T1 is very short.
