TEAR PROTEIN-G ORIGINATES FROM DENATURED TEAR SPECIFIC PREALBUMIN AS REVEALED BY 2-DIMENSIONAL ELECTROPHORESIS

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (ID-SDS PAGE) of the non-denatured low molecular weight (MW) tear proteins (dilution in phosphate buffered saline or in the non-ionic detergent Triton X 100) revealed no protein G but a strongly marked 23-kD related to a tear specific prealbumin (TSP) subunit coming with the known 15, 17, 18 and 20-kD TSP subunits. Under mild denaturating conditions of sample preparation with SDS dilution just before electrophoresis, 23-kD protein decreased and a faint 32-kD protein G appeared. Under stronger denaturing conditions of sample preparation with SDS treatment (boiling or freeze-thaw cycles), 23-kD protein disappeared and two main protein G forms (32 and 34-kD) and additional bands (29, 36, 39, 42, 57 and 60-kD) appeared depending on the sample treatment. The isoelectric pH (pI) of these proteins ranged from pH 5.2 to pH 5.4. Different two-dimensional electrophoresis methods revealed that: - in presence of SDS, 23-kD protein was spontaneously changed into 17-kD TSP and such a phenomenon was partially reversible by using a non-ionic detergent (Triton X100), - new proteins appeared under denaturating processes were related to various protein G forms and originated from TSP group, - proteins G were produced by the aggregation of TSP subunit related to MW 17-kD/pI 5.0 corresponding to the major TSP subunit, - disulfide bond formation was shown to play a major role in the aggregation process although protein G group was not totally reduced by dithiotreitol. Such results suggest that protein G is an in vitro experimental artifact due to denaturing conditions with SDS treatment. Protein G originates in the aggregation of 17-kD TSP partly formed by denaturation of 23-kD TSP.