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Studies of fluorescence lifetimes and quenching of fluorescence by iodide ions introduced into the endoplasmic reticulum lumen reveal that the signal sequence and nascent chain of secretory proteins are inaccessible to ions on the cytoplasmic side of the membrane and are located in an aqueous space separated from the cytoplasm and lumen. Translocation of the nascent chain occurs only when the chain length exceeds 70 amino acids.

The signal sequence which plays a major role in the early stages of the translocation process of a nascent secretory protein across the endoplasmic reticulum (ER) membrane is located at an aqueous space or compartment formed by the tight binding of the ribosome to the ER membrane. The signal sequence and the nascent protein chain pass through a tunnel to reach the ER membrane. The iodide ions are prevented from entering the nascent chain pathway due to the permeability barrier between cytoplasm and the ER.

Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.