Tear protein G originates from denatured tear specific prealbumin as revealed by two-dimensional electrophoresis.
One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS PAGE) of the non-denatured low molecular weight (MW) tear proteins (dilution in phosphate buffered saline or in the non-ionic detergent Triton X100) 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 dithiothreitol. Such results suggest that protein G is an in vitro experimental artifact due to denaturing conditions with SDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Baguet
J
J
Laboratoire Meuse Optique Contact (MOC), Centre Hospitalier, Bar le Duc, France.
Claudon-Eyl
V
V
Gachon
A M
AM
D000328:Adult; D016477:Artifacts; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000032:analysis*; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis*; D006801:Humans; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D011228:Prealbumin / Q000737:chemistry*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013666:Tears / Q000737:chemistry*
D005136:Eye Proteins; D011228:Prealbumin; C042434:tear proteins; D012967:Sodium Dodecyl Sulfate; D019204:GTP-Binding Proteins