A comparison of the spin labels MAL-3 and TEMPAMINE for measuring the internal microviscosity of human erythrocytes.

In a recent paper, Daveloose et al. (Daveloose, D., Fabre, G., Berleur, F., Testylier, G. and Letterrier, F. (1983) Biochim. Biophys. Acta 763, 41-49) described a technique to measure the internal microviscosity of erythrocytes using the spin label MAL-3 (2,2,5,5-tetramethyl-3-malimidopyrrolidinyl-N-oxyl) to supercede the use of TEMPAMINE (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine)ferricyanide. They cite the long time (12 h) required for TEMPAMINE to enter the erythrocyte interior and the 'unphysiological' nature of the potassium ferricyanide required to isolate the TEMPAMINE signal inside the erythrocytes as reasons for developing this technique. In the present work, the penetration of TEMPAMINE into the erythrocyte interior is found to be pH-dependent and on the order of seconds, not hours. The slow penetration of TEMPAMINE described by Daveloose et al. probably is a result of their using trimethylTEMPAMINE which is membrane-impermeable. High concentrations of ferricyanide do affect red cell morphology in a reproducible fashion. The internal microviscosity as measured by TEMPAMINE reflects this. MAL-3 does not. Sample preparation time using TEMPAMINE-ferricyanide is about 1 min compared to about 40 min for the MAL-3 technique. In the presence of ferricyanide or ascorbate, MAL-3-labeled cells show slow loss of signal intensity which indicates that MAL-3 leaks out of the cells. Both the TEMPAMINE-ferricyanide and MAL-3 techniques have advantages and disadvantages.