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Identification of lactoferrin in bovine tears
M H Brown1, A H Brightman, B W Fenwick
1Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan 66506-5606, USA.
Objective:
To determine whether bovine tear film contains the iron-binding glycoprotein, lactoferrin.
Animals:
40 Adult Hereford, Angus, and Simmental cattle.
Procedure:
Protein analysis: pooled bovine tears were used for protein analysis (size exclusion high-performance liquid chromatography [HPLC] fractionation). HPLC was used for tear analysis. A diode array detector was used (215 and 280 microns) for chromatogram analysis and comparisons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): protein electrophoresis was performed, using 7.5% running gels with 4% stacking gels. Molecular weight of proteins in the unknown samples was determined as recommended by the manufacturer of the standards. Protein sequencing: amino acid sequencing, using automated Edman degradation of HPLC purified protein, was performed. The sequence 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 nearly identical to purified bovine lactoferrin. Silver-stained SDS-PAGE of this peak revealed a band with molecular mass consistent with bovine lactoferrin (estimated mass of 78 kd). The first 13 amino acid residues of this protein were identical to the amino acid sequence of bovine 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 from potential pathogens.