Physicochemical characterization of psychosine by 1H nuclear magnetic resonance and electron microscopy

Krabbe's disease is an autosomal recessive disease that affects the lysosomal enzyme galactosylceramidase. The storage of one of its substrates, psychosine (beta-galactosyl-sphingosine), is thought to be responsible for the induction of pathological changes. Psychosine has a free amine group which is necessary for the mediation of its toxic effects. In the present study, the physicochemical properties of psychosine were investigated. Nuclear magnetic resonance (NMR) detected pH titration was used to determine that the amine group had a pKa of 7.18 +/- 0.05. Pulsed-field gradient NMR spectroscopy was used to determine that the diffusion coefficient of 2.8 mM psychosine in D2O at pD 4.46 or 7.04 is 1.16 +/- 0.02 x 10(-10) m2/s or 0.77 +/- 0.02 x 10(-10) m2/s, respectively. Negative staining electron microscopy (EM) studies of acidic and neutral solutions of psychosine also were performed. At pH 4.5, spherical structures were formed, which were relatively stable between 3, 120, and 216 h following preparation; the diameter ranged from approximately 14 nm at the earliest time point to approximately 18 nm at the last time point. The critical micelle concentration (CMC) was 1.26 mM at pH 4.0. At pH 7.1, the structures changed from spherical structures with a diameter of 15-23 nm, at the earliest time point, to a heterogeneous population of structures ranging from spherical structures, with a diameter of only a few nm, to irregularly shaped oblong structures that had one or more dimensions exceeding 100 nm. The NMR and EM data indicate that the deprotonation of the amine group causes psychosine to form aggregates that are unstable, which prevents a determination of the CMC at a neutral pH. These data indicate that molecular interactions of psychosine at the acidic pH of the lysosome, where it is normally digested, are more orderly than those at the pH of the cytoplasm or extracellular space where psychosine goes during disease.