PHOSPHORIC-ACID ENTRAPMENT LEADS TO APPARENT PROTEIN HETEROGENEITY

Recombinant proteins produced in prokaryotes or eukaryotes show certai n types of heterogeneity due to post-translational modifications. Some preparations of a soluble interferon gamma receptor, produced in Esch erichia coli, appeared as a double band with slightly different mobili ties in non-reducing sodium dodecylsulfate and native polyacrylamide g els. Ion spray mass spectrometry showed that the two forms had a mass difference of one to three multiples of 97 +/- 2 D. Gas chromatography -mass spectrometry analysis revealed the presence of phosphoric acid i n the hydrolysate and in the intact protein. The more slowly migrating protein species had trapped molecules of phosphoric acid during the p rotein extraction. Most of the trapped phosphoric acid was loosely ass ociated with the protein. One to three molecules were tightly, but non -covalently linked per receptor molecule. Phosphoric acid entrapment d id not affect biological activity and most likely did not affect prote in conformation. The species carrying phosphoric acid showed higher so lubility, Trapping of phosphoric acid by proteins may be a general phe nomenon and the results reported here thus useful in the characterizat ion of other recombinant proteins.