BINDING CONSTANTS FOR A PHYSIOLOGICAL ELECTRON-TRANSFER PROTEIN COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN - EFFECTS OF IONIC-STRENGTH AND BOUND COPPER ON BINDING
Vl. Davidson et al., BINDING CONSTANTS FOR A PHYSIOLOGICAL ELECTRON-TRANSFER PROTEIN COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN - EFFECTS OF IONIC-STRENGTH AND BOUND COPPER ON BINDING, Biochimica et biophysica acta, 1144(1), 1993, pp. 39-45
Two soluble proteins, methylamine dehydrogenase and amicyanin, form a
physiologically relevant complex in which intermolecular electron tran
sfer occurs. To characterize and quantitate the binding of these two w
eakly-associating proteins, an ultrafiltration binding assay has been
developed which involves brief centrifugation of mixtures of proteins
in centrifuge concentrators followed by quantitation of proteins on ea
ch side of the filtration membrane by HPLC. Under low ionic strength c
onditions which are optimal for the redox reaction between these prote
ins, a K(d) of 4.5 muM was measured for the methylamine dehydrogenase-
amicyanin complex. The K(d) increased by 8-fold in the presence of add
ed salt. Apoamicyanin, which is known from crystallographic analysis t
o be structurally very similar to amicyanin, exhibited a much higher K
(d) and much less specific binding than did the holoprotein. Apoamicya
nin also exhibited apparent self-association at low ionic strength whi
ch was not observed with amicyanin. These observations are correlated
with the known crystal structures of these proteins, free and in compl
ex, and with the available biochemical information on the interactions
of these two proteins.