IDENTIFICATION OF LACTOFERRIN IN BOVINE TEARS

Objective-To determine whether bovine tear film contains the iron-bind ing glycoprotein, lactoferrin. Animals-40 Adult Hereford, Angus, and S immental cattle. Procedure-Protein analysis. pooled bovine tears were used for protein analysis (size exclusion high-performance liquid chro matography [HPLC] fractionation). HPLC was used for tear analysis. A d iode array detector was used (215 and 280 mu m) for chromatogram analy sis and comparisons. Sodium dodecyl sulfate-polyacrylamide gel electro phoresis (SDS-PAGE): protein electrophoresis was performed, using 7.5% running gels with 4% stacking gels. Molecular weight of proteins in t he unknown samples was determined as recommended by the manufacturer o f the standards. Protein sequencing: amino add sequencing, using autom ated Edman degradation of HPLC purified protein, was performed. The se quence obtained was compared with the known protein sequence of bovine lactoferrin. Results-HPLC analysis of whole bovine tears resulted in a consistent chromatogram. Peak collection was performed to recover a protein from the bovine tear film with chromatogram characteristics ne arly identical to purified bovine lactoferrin. Sliver-stained SDS-PAGE of this peak revealed a band with molecular mass consistent with bovi ne lactoferrin (estimated mass of 78 kd). The first 13 amino acid resi dues of this protein were identical to the amino acid sequence of bovi ne lactoferrin. Conclusion-Analysis of whole bovine tears, using size exclusion HPLC, SDS-PAGE, and amino acid sequencing, provided evidence that bovine tears contain lactoferrin. Clinical Relevance-Lactoferrin probably exerts a bacteriostatic effect in bovine tear film. Locally produced lactoferrin may bathe the ocular surface and sequester iron f rom potential pathogens.