Gel electrophoresis of human tears reveals various forms of tear lactoferrin.
Lactoferrin is a metal binding protein, which is present in high concentrations in human tears. Little is known concerning the exact molecular shape of lactoferrin in tears. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting experiments showed that this protein is present in multiple forms in tear fluid. SDS-PAGE analysis of human tears under non reducing conditions and pretreatment of tears in sample buffer at room temperature revealed lactoferrin in a major form of 60 kD, a minor form of 64 kD and a third form of 52 kD. Pretreatment of tears at elevated temperatures prior to sample application resulted in the loss of this third form. Disruption of intrachain disulfide bridges prior to SDS-PAGE analysis resulted in a shift in the apparent molecular weight of lactoferrin to 78 kD and 83 kD for the major and minor form, respectively. Chromatography of human tears on ConA-Sepharose as well as enzymatic deglycosylation showed that the difference in molecular weight of the major and minor lactoferrin form was not due to a variation in the carbohydrate side chains. The presence of the minor form could also not be ascribed to iron saturation. Instead we found that addition of iron ions to human tears resulted in a shift of tear lactoferrin to a lower molecular weight species of about 52 kD, coinciding with the third lactoferrin form mentioned above and a small protein band of approximately 57 kD, representing the iron saturated minor lactoferrin form. Similar findings were observed using purified milk lactoferrin. Increasing the temperature prior to sample application or disruption of disulfide bridges dissociated the iron-lactoferrin complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Kijlstra
A
A
Department of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, Amsterdam.
Kuizenga
A
A
van der Velde
M
M
van Haeringen
N J
NJ
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D007502:Iron Chelating Agents / Q000378:metabolism; D007781:Lactoferrin / Q000032:analysis*; D007782:Lactoglobulins / Q000032:analysis*; D008892:Milk / Q000032:analysis*; D008970:Molecular Weight; D013666:Tears / Q000032:analysis*; D013696:Temperature
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007502:Iron Chelating Agents; D007782:Lactoglobulins; D007781:Lactoferrin