Study of subunit interactions of alpha A- and alpha B-crystallins and the effects of gamma-irradiation on their interactions by surface plasmon resonance

Alpha-crystallin, a major protein of mammalian lens, consists of two subunits, alpha A-crystallin and alpha B-crystallin. They interact to form an aggregate and play a prominent role in the maintenance of lens transparency. We evaluated the interaction between these subunits via surface plasmon resonance (SPR) using four combinations of immobilized protein and analyte: 1) AA: alpha A-crystallin was ligand immobilized onto the sensor and alpha A-crystallin was passed over the ligand, 2) AB: ligand - alpha A-crystallin, analyte - alpha B-crystallin, 3) BB: ligand - alpha B-crystallin, analyte- alpha B-crystallin, 4) BA: ligand - alpha B-crystallin, analyte - alpha A-crystallin. The order of rate of dissociation was AA approximately BA>BB approximately AB. We also examined the dissociation of gamma irradiated alpha A- and alpha B-crystallins. As radiation dose increased, so did the dissociation rate of all of the crystallins. The order of rate of dissociation of irradiated crystallins was BB>AB approximately BA>AA. The results indicate that BB is the most susceptible to gamma-irradiation and that alpha B-crystallin forms a more stable aggregate than alpha A-crystallin under normal conditions. However, when alpha B is irradiated the aggregate becomes unstable.     

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components—ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)—suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.     

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.     

Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

ρB-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens ρ-crystallin. We have now determined the complete cDNA sequence of ρB-crystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog ρ-crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, ρB-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys → Tyr, ρB-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the ρB-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant ρB-crystallin nor the reverse mutant 298 Tyr → Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; ρB-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end. Received: 18 July 2000 / Accepted: 3 November 2000     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

Dietary γ-aminobutyric acid affects the brain protein synthesis rate in young rats

Summary. The purpose of this study was to determine whether the γ-aminobutyric acid (GABA) affects the rate of brain protein synthesis in male rats. Two experiments were done on five or three groups of young rats (5 wk) given the diets containing 20% casein administrated 0 mg, 25 mg, 50 mg, 100 mg or 200 mg/100 g body weight GABA dissolved in saline by oral gavage for 1 day (d) (Experiment 1), and given the diets contained 0%, 0.25% or 0.5% GABA added to the 20% casein diet (Experiment 2) for 10 d. The plasma concentration of growth hormone (GH) was the highest in rats administrated 50 mg and 100 mg/100 g body weight GABA. The concentration of serum GABA increased significantly with the supplementation groups. The fractional (Ks) rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein + 0.25% GABA diet and still more 20% casein + 0.5% GABA compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. Our results suggest that the treatment of GABA to young male rats are likely to increase the concentrations of plasma GH and the rate of protein synthesis in the brain, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Trehalose effects on alpha-crystallin aggregates

alpha-Crystallin in its native state is a large, heterogeneous, low-molecular weight (LMW) aggregate that under certain conditions may progressively became part of insoluble high-molecular weight (HMW) systems. These systems are supposed to play a relevant role in eye lens opacification and vision impairment. In this paper, we report the effects of trehalose on alpha-crystallin aggregates. The role of trehalose in alpha-crystallin stress tolerance, chaperone activity and thermal stability is studied. The results show that trehalose stabilizes the alpha-crystallin native structure, inhibits alpha-crystallin aggregation, and disaggregates preformed LMW systems not affecting its chaperone activity.     

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H₂O₂ on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H₂O₂ and 0.1 mM FeCl₃ at 37 ^∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.     

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.     

Diuretic potential of energy drinks

Summary. Recent literature suggests that both caffeine and taurine can induce diuresis and natriuresis in rat and man. Although they act via different cellular mechanisms, their diuretic actions might be additive. This is of considerable interest, as several commercially available energy drinks contain both substances. In this study we examined the possible diuretic effects of caffeine and taurine in a cross-over-design in which 12 healthy male volunteers received each of 4 different test drinks (750 ml of energy drink containing 240 mg caffeine and 3 g taurine, the three other test drinks either lacked caffeine, taurine or both) after restraining from fluids for 12 h. Mixed model analyses demonstrated that urinary output and natriuresis were significantly increased by caffeine (mean differences 243 ml and 27 mmol; both p < 0.001) and that there were no such effects of taurine (mean differences 59 ml and −4 mmol). Additionally, urinary osmolarity at baseline was significantly related to the urinary output (p < 0.001). Urine osmolarity values at baseline and in the 6 h urine collection did not differ significantly between treatments. Taken together, our study demonstrates that diuretic and natriuretic effects of the tested energy drink were largely mediated by caffeine. Taurine played no significant role in the fluid balance in moderately dehydrated healthy young consumers. Consequently, the diuretic potential of energy drinks will not differ significantly from other caffeine containing beverages.     

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.     

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism

 The negative gravitropism of the sporangiophores of Phycomyces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132–142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15–25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains. Received: 2 September 1999 / Accepted: 9 November 1999     

Nitric oxide is involved in taurine release in the mouse brain stem under normal and ischemic conditions

Summary. Nitric oxide (NO) has been shown to regulate neurotransmitter release in the brain; both inhibitory and excitatory effects have been seen. Taurine is essential for the development and survival of neural cells and protects them under cell-damaging conditions. In the brain stem, it regulates many vital functions such as cardiovascular control and arterial blood pressure. Now we studied the effects of the NO-generating compounds hydroxylamine (HA), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) on the release of preloaded [³H]taurine under normal and ischemic conditions in slices prepared from the mouse brain stem from developing (7-day-old) to young adult (3-month-old) mice. In general, the effects of NO on the release were somewhat complex and difficult to explain, as expected from the multifunctional role of NO in the central nervous system. The basal initial release under normal conditions was enhanced by the NO donors 5 mM HA and 1.0 mM SNAP at both ages, but SNP was inhibitory in developing mice. The release was markedly enhanced by K⁺ stimulation. The effects of HA, SNAP and SNP on the basal release were not antagonized by the NO synthase inhibitor N^G-nitro-L-arginine (L-NNA, 1.0 mM), demonstrating that mechanisms other than NO synthesis are involved. Taurine release in developing mice in the presence of SNP was reduced by the inhibitor of soluble guanylate cyclase, 1H-(1,2,3)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), indicating the possible involvement of cGMP. In normoxia, N-methyl-D-aspartate (NMDA, 1.0 mM) enhanced the SNAP- and HA-evoked taurine release in developing mice and the HA-evoked release in adults. In ischemia, both K⁺ stimulation and NMDA potentiated the NO-induced release, particularly in the immature mice, probably without the involvement of the NO synthase or cGMP. The substantial release of taurine in the developing brain stem evoked by NO donors together with NMDA might represent signs of important mechanisms against excitotoxicity which protect the brain stem under cell-damaging conditions. Authors’ address: Prof. Pirjo Saransaari, Brain Research Center, Medical School University of Tampere, Tampere, FIN-3 3014, Finland     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

Free glutamine as a major precursor of brown products and fluorophores in Maillard reaction systems

Summary. Glutamine is one of the most abundant free amino acid found in raw food. In this study, the contribution of free glutamine to nonenzymatic browning and fluorescence was investigated using an aqueous model system with methylglyoxal. The results indicated that glutamine contributed to the Maillard reaction via two pathways. First, the hydrolysis of the amide bond of glutamine led to the release of ammonia which was implicated in the formation of brown color and fluorescence. Among other nitrogen donors tested (asparagine, glutamic acid and urea) our results demonstrated that free glutamine was a major source of ammonia during heating. When heated at 120 and 180 °C, 100% of ammonia was released from glutamine after 60 and 10 min, respectively. The second pathway involved a direct Maillard reaction with the α-amino group of glutamine. Both pathways led to a rapid and complete destruction of glutamine when heated in the model systems. With reference to the Maillard browning (absorbance at 420 nm) glutamine turned out to be the most reactive amine, followed by asparagine, glutamate, ammonia and urea. Maximum fluorescence (excitation and emission wavelengths at 330 and 450 nm, respectively) was also observed with glutamine followed by urea and ammonia. Overall this study suggested that free glutamine predominantly contributes to the color and fluorescence formations of foodstuffs.     

Asp isomerization increases aggregation of α-crystallin and decreases its chaperone activity in human lens of various ages

α-Crystallin, comprising 40–50 subunits of αA- and αB-subunits, is a long-lived major soluble chaperone protein in lens. During aging, α-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in α-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of αA-crystallin by a series of analyses of the soluble α-crystallin, HMW and WI fractions from human lens samples of different ages (10–76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10–450 S) for lens of all ages, whereas the α-crystallin had a much smaller peak sedimentation coefficient (10–20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (Lα-, Lβ-, Dα-, Dβ-) at Asp58, Asp91/92 and Asp151 in αA-crystallin by liquid chromatography–mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (α-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, Dβ-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of α-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of α-crystallin in aged human lens.     

Convenient synthesis of GOLD and MOLD and identification of their oxidation products in vitro and in vivo

Summary. Two Lys–Lys crosslinks, 1,3-bis-(5-amino-5-carboxypentyl)-1H-imidazolium (GOLD) and 1,3-bis(5-amino-5-carboxypentyl)-4-methyl-1H-imidazolium (MOLD) salts, have been synthesized by the reaction of imidazole or 4(5)-methyl imidazole with 5-(4-bromobutyl)-hydantoin followed by the hydrolysis of 1,3-substituted imidazolium derivatives by 6.0 N HCL at 110 °C. Treatment of GOLD and MOLD with hydrogen peroxide in acetic acid leads to MOLD oxidation only. The oxidation product of MOLD was detected in cataractous lens proteins.     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.