Application of solid‐phase extraction for the concentration of chromophores, fluorophores, and photosensitizers from lens protein digests

• Published in: Journal of separation science Vol. 37; no. 21; pp. 3106 - 3109
• Main Authors: Argirov, Ognyan K, Hubenova, Yolina, Argirova, Mariana D
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH 01.11.2014; Blackwell Publishing Ltd; Wiley; Wiley Subscription Services, Inc
• Subjects: absorption; Animals; Biological and medical sciences; calves; Cataracts; Cattle; Chemical compounds; Chromophores; Crystallins - chemistry; Crystallins - isolation & purification; Dehydroascorbic acid; Digestion; Extraction; Extraction processes; eye lens; Eyes & eyesight; Fluorescence; Glycation; Glycosylation; Human; Humans; irradiation; Lens diseases; Lens proteins; Lens, Crystalline - chemistry; Lenses; Medical sciences; Molecular chemistry; Ophthalmology; photosensitivity disorders; Proteins; Separation; singlet oxygen; Solid Phase Extraction - methods; Solid-phase extraction; solvents; ultraviolet radiation; Cataracts; Lens proteins; Solid phase extraction; Peptides; Glycation; Chemical enrichment; Eye; Separation method; Sample preparation; Chromophore; Trace analysis; Human; Glycated protein; Cataract; Fluorescence detector; Healthy subject; Patient; Protein; Solid-phase extraction; Eye disease; Dehydroascorbic acid; Lens disease; Ultraviolet detector; Fluorophore; Peptide fragment; Lens; Photosensitizer; Anterior segment disease
• ISSN: 1615-9306; 1615-9314; 1615-9314
• DOI: 10.1002/jssc.201400285

Solid‐phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid‐phase extraction was capable of concentrating the advanced glycation end‐products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.