Abstract
Purpose: :
Protein-protein interactions may play a role in tear film stability due to their effect on tear film viscosity. Metal cations in solution have an effect on protein-protein interactions and thus tear film stability. As rabbits have a more stable tear film than humans, we sought to examine the tears of rabbits and humans for protein-protein interactions, metal types and concentrations and how they affect protein-protein interactions.
Methods: :
Tears were collected from normal adult humans (n=10) and rabbits (n=6) using microcapillary tubes and pooled for each study group. Tear metal cations were analyzed using inductively coupled plasma (ICP) mass spectrometry and ICP atomic emission spectroscopy. Protein-protein interactions were studied using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The effect of cations on protein-protein interactions was studied by using modified BN-PAGE in which cations were added to sample buffer and running buffer at levels of 0, human tears or rabbit tears.
Results: :
Major metals in tears did not differ between species, however, rabbit tears had a higher concentration of divalent cations: [Mg2+], Human=0.39mM, Rabbit=1.13mM; [Ca2+], Human=0.38mM, Rabbit=0.75mM. In the absence of cations, protein-protein interactions were identified in both human (DMBT1-lactoferrin-lysozyme and lactoferrin-lipocalin) and rabbit tears (interactions of lipophilin subclasses). Additional protein-protein interactions were found in rabbit tears (prolactin inducible protein-lipophilins), but not in human tears, in the presence of cations at either of concentrations tested.
Conclusions: :
The presence of cations shows no effect on protein-protein interactions in human tears, but increases the protein-protein interactions in rabbit tears. However, the cation’s contribution to tear film stability needs further investigation.
Keywords: cornea: tears/tear film/dry eye • protein structure/function • proteomics