Gel electrophoresis of human tears reveals various forms of tear lactoferrin

Abstract

Lactoferrin is a metal binding protdn, which is present in high concentrations in human rears. Little is known concerning the exact moleclllar shape of lactoferrin in tears. Sodium dodecyl sdphate 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 lactofemn 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 lactofenin. Increasing the temperature prior to sample application or disruption of disulfide bridges dissociated the iron-lactoferrin complex.

These experiments indicate that the minor lactoferrin form is a larger peptide than the major form and may represent a lactoferrin precursor. Furthermore this report is the first showing the presence of iron saturated lactoferrin in human tears, which may have been missed by earlier investigations due to high temperature treatment or reduction of samples. The techniques described in this report may provide further insight in the role of this iron binding protein on the ocular mucosa in health and disease.