Purpose  To demonstrate the presence of hemoglobin (Hb) in human lens capsule epithelium, aqueous humor, and pseudoexfoliation material (PXFM).
Methods  Lens capsules (LC) and aqueous humor (AH) were obtained from normal and pseudoexfoliation (PXF) eyes during cataract extraction surgery through avascular cornea and without contamination by hemorrhage. Proteomic analysis of pooled aqueous humor and lens capsule was performed using electrospray tandem mass spectrometry. Immunofluorescence (IF) was used to localize Hb within lens capsules and PXFM on LC, which is pathognomonic for PXF. Western blotting of solubilized LC (pooled by minimum 7 specimens) and AH (concentrated after speed vac) was used to compare Hb expression levels between normal and PXF eyes. Len capsules were also stained with hematoxylin/eosin and Prussian blue (to detect iron).
Results  Proteomic analysis of LC and AH differentially identified the presence of Hb in eyes with PXF compared to normal eyes. Immunofluorescence localized Hb to PXF material and lens capsule epithelium. Western blot analysis demonstrated a ~14 kDa Hb protein band in both normal and PXF LC and AH that was significantly greater in PXF eyes. Prussian blue staining was negative suggesting the lack of iron-chelated Hb. 
Conclusions  Using proteomic analysis, immunofluorescence, and immunoblotting, non-iron-chelated hemoglobin was observed to be highly expressed in avascular human LC, PXFM, and AH. Prussian blue negative staining of Hb suggests no contamination from bleeding during surgical specimen extraction. Over a decade ago, several groups suggested a low molecular weight protein was associated with PXF glaucoma. We have identified this molecule to be Hb, which is specifically present within PXF material.