Isoelectric focusing studies of bacteriorhodopsin.

Purified bacteriorhodopsin (BR) samples show a minimum of four isoelectric forms in immobilized pH gradient isoelectric focusing gels. The bands occur as doublets with isoelectric points (pI) centered at 5.20 (principal species) and 5.60. In typical preparations additional bands may be observed at 4.90, 5.07 and 5.50. Purple membrane (PM) was proteolyzed with papain to calibrate the pI shift produced by changing the number of charges on the protein. Asp-242 is removed during the first cleavage between residues 239 and 240 resulting in the loss of a single negative charge and a shift of the principal doublet by +0.35 pH units to pI 5.55. The second papain cleavage occurs between residues 231 and 232 which removes Glu-232, -234 and -237 and shifts the pI by +0.60 pH units to pI 6.10. The +0.60 pH shift upon the second papain cleavage is consistent with the loss of two negative charges and is supported by prior evidence that at least one of the three glutamate residues lost during the second proteolysis step is protonated and neutral in the intact protein. The native and proteolyzed products of BR retain the characteristic 550 nm absorption maxima for solubilized BR. A model for the structural origin of the pI heterogeneity of BR species in proteolyzed PM is presented.