Purpose: Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, [beta]A3/ A1102-117 peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by [beta]- and [gamma]-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of [alpha][beta]-crystallin. The present study was performed to identify the interacting sites of the [beta]A3/A1102-117 peptide in [alpha]B-crystallin. Methods: [beta]A3/A1102-117 peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{pazidobenzamido}- hexanoamido] ethyl-1-3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotincontaining cross-linker. The biotin-derivatized peptide was then incubated with [alpha]B-crystallin at 37 [degrees]C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to [alpha]B-crystallin. Label transfer was confirmed by western blot, and the labeled [alpha]B-crystallin was digested with trypsin. Tryptic peptides from [alpha]B-crystallin carrying the biotin label were purified by avidin affinity chromatography, and [beta]A3/A1102-117 peptide interacting sites in [alpha]B-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS). Results: We found that the [beta]A3/A1102-117 peptide interacted with [alpha]B-crystallin regions 70LEKDR74, 83HFSPEELKVK92, 91VKVLGDVIEVHGK103, 93VLGDVIEVHGKHEER107, and 121KYR123, which are part of the [alpha]-crystallin domain, and were previously shown to be part of the functional chaperone site in [alpha]B-crystallin. The [beta]A3/A1102-117 peptide also interacted with regions at the COOH-terminal extension of [alpha]B-crystallin, 150KQVSGPER157, 164EEKPAVTAAPK174, and 164EEKPAVTAAPKK175. When two of the hydrophobic residues of [beta]A3/A1102-117 peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property. Conclusions: This study confirmed the interactions between a low molecular weight peptide derived from [beta]A3/A1- crystallin found in aged and cataract lenses and [alpha]B-crystallin. The binding of [beta]A3/A1102-117 peptide to the chaperone site and the COOH-terminal extension of [alpha]B-crystallin may explain its anti-chaperone property
