Binding of 1‐Anilinonaphthalene‐8‐Sulfonic Acid to α‐crystallin

Abstract

a-Crystallin was found to exhibit a time-dependent uptake of the hydrophobic probe, l-anilinonaphthalene-8-sulfonic acid (ANS), similar to that typically observed with lipid membranes. Analysis of the interaction of ANS with a-crystallin revealed two types of interactive processes, partitioning and binding. The predominant process involved partitioning, with a coefficient of 300 M-'. The binding component had the following characteristics: 1 binding site124 subunits and a Kd of about 9 pM. The binding was unaffected by the number of subunits used in the assembly of the a-aggregate, since both the an,-and a,-forms had similar binding characteristics. No discernible differences were observed in the binding of ANS to homopolymers of aA and aB subunits, suggesting that the hydrophobic sites to which ANS bound were similar in both the A and B subunits. The majority of the fluorescence was lost when the protein was incubated in 3 M urea, a concentration of denaturant where the protein is still intact, suggesting that the ANS binding sites are located near the surface of the protein. The decrease was attributed to a decrease in the quantum yield of the bound dye.Keywords: a-crystallin; I-anilinonaphthalene-8-sulfonic acid; quaternary structure; hydrophobic interaction.a-Crystallin constitutes up to 50% of the total proteins which comprise the bulk of the mammalian eye lens. Its relatively constant proportion in most species suggests that it has an important role in the lens. The absence of any tissue turnover necessitates that it remains functional for the lifetime of the organism, in the case of humans in excess of 70 years. In cases where the protein is modified or structurally disrupted impairment of vision results, usually in the form of a cataract. Recently, it has been shown that a-crystallin is a member of the small heat-shockprotein family and may function as a chaperone [I]. It has been suggested that one of its major functions is to prevent age-related aggregation of lens proteins, thereby maintaining lens transparency [2]. The mechanisms for this function have not been delineated but they probably involve the surface of a-crystallin which has been shown to be very hydrophobic [3, 41. Knowledge of the location of these hydrophobic sites would be of enormous benefit in determining how a-crystallin functions as a molecular chaperone, as well as providing insight into its structure.a-Crystallin is a polymeric protein which consists of two types of subunits, A and B, both of which have a mass around 20 kDa [5]. Their amino acid sequences are closely related and have changed very slowly during the course of evolution, reflecting a structural constraint on the protein. This also suggests that it has an important role in maintaining interactions necessary for the formation of a transparent lens. Both polypeptides are subject to various chemical and enzymatic modifications such as oxidation, phosphorylation, deamidation, glycation, and C-terminal degradation 161. These modifications are thought to

Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins

“…These data suggest that hydrophobic forces are dominant in the ANS binding to ␣-crystallin. The fact that ␣A-and ␣B-crystallin have two sets of binding sites is in agreement with the studies of Stevens and Augusteyn, who suggested two kinds of interactions for the binding of ANS to ␣-crystallin (22). Moreover, binding of destabilized mutants of T4 lysozyme to ␣-crystallin was demonstrated to be biphasic (28).…”

“…Historically, hydrophobicity has been measured qualitatively and quantitatively by spectroscopic methods like fluorescence and equilibrium dialysis, mostly by using two common fluorescent dyes, 8-anilino-1-naphthalenesulfonic acid (ANS) and bis-ANS (19,20,22,24). In such cases, indirect methods such as van't Hoff analysis for obtaining thermodynamic parameters to elucidate ANS binding are often limited.…”

Interaction of 1,1′-Bi(4-anilino)naphthalene-5,5′-Disulfonic Acid with α-Crystallin

“…The difference in dissociation constants determined by the three methods was not significant. These values are five to eight times lower than the K d value for ANS binding to ␣-crystallin reported recently (39).…”

“…There is, however, disagreement with respect to the number of hydrophobic sites available within ␣-crystallin as determined by the environment-sensitive probe ANS (38,39). We used bis-ANS instead of ANS to determine the nature of hydrophobic sites in ␣-crystallin.…”

“…Liang and Li (38) reported that there are about 40 ANS binding sites/native ␣-crystallin. Stevens and Augusteyn (39) have disputed the study of Liang and Li and reported that there is one ANS binding site/24 subunits of ␣-crystallins. It is rather difficult to explain the stoichiometry of ANS binding to ␣-crystallin in view of the proposed complex but ordered structure for ␣-crystallin (2).…”