In the first year of this grant, we have demonstrated that 32P-spectrin tetramers bind to spectrin-depleted inverted vesicles with equal affinity to spectrin heterodimers (K equals 17 nM) at physiological ionic strength, pH 7.6, 4 degrees C. Close to the physiological amount of spectrin could be rebound to spectrin depleted inverted vesicles when added as a tetramer, while only one-half maximal capacity could be obtained with the heterodimer. We concluded that the tetramer is the physiological state of the spectrin molecule, and the stoichiometry in vivo is 1 spectrin tetramer/syndein attachment site, (Goodman and Weidner, JBC, 255: 8082-8086, 1980). Since spectrin heterodimers and tetramers rebind to the erythrocyte membrane with equal affinity, either species could be used to compare the spectrin-membrane interaction in normal and abnormal erythrocytes. We have found that the protein composition of HS red cell ghosts, spectrin depleted inverted vesicles, and spectrin heterodimers is quantitatively normal in 9 HS patients from 3 unrelated families under our study. The binding of spectrin heterodimers to spectrin depleted inverted vesicles indicated a KD of 18 plus or minus 2 nM and a maximal binding capacity of 98 plus or minus 7 micrograms of spectrin bound/mg membrane protein for 9 HS patients and 18 plus or minus 2 nM and 116 plus or minus 8 micrograms spectrin/mg membrane protein in 9 paired normal controls. Therefore the binding of spectrin to the syndeins (bands 2.1-2.6) its high affinity membrane binding site is unaltered in hereditary spherocytosis. If the primary alteration in hereditary spherocytosis resides in the spectrin membrane skeleton as previously suggested (Lux, 1979), then our results suggest that our search must now be focused on the spectrin-4.1-actin and syndein-band 3 interactions. We plan to compare these interactions in HS versus normal erythrocytes by (1) studying the ability of band 4.1 and actin to bind to purified spectrin in solution, (2) studying the ability of purified band 2.1 (syndein) to bind to KI stripped vesicles (in which the band 3 binding sites are accessible), and (3) one and two dimensional peptide mapping of spectrin, syndein, band 3, band 4.1 and actin.