Raymond F. Borkman

Spectroscopic Evaluation and Classification of the Normal, Aging, and Cataractous Lens. (With 1 color plate)

Fluorescence, UV and visible and EPR spectroscopy studies were performed on normal human lenses ranging in age from 1 day to 92 years and on various cataractous lenses. These experiments revealed two

ULTRAVIOLET RADIATION IN THE AGING AND CATARACTOUS LENS: A Survey*

An increase in the insoluble protein levels, in fluorescence, and a decrease in the SH concentration are three specific aging parameters in the mammalian lens. These can be markedly accelerated in human, rat and mouse lenses by in vitro exposure (for at least 4 h) to UV radiation (above 300 nm) and 3‐aminotriazole (AT) and in vivo by exposing mice to UV and parenteral AT for 4–6 weeks. UV, fluorescence, electron paramagnetic resonance (EPR) and Lasar Raman Spectroscopy were performed on normal and UV plus AT exposed 4–6 week old rat lenses and normal and experimental human lenses (1 day to 75 years of age). These data demonstrate the presence and/or induction of at least one fluorescent compound (fluorogen) (360 nm activation, 440 nm emission) derived from tryptophan by a free radical induced photooxidation reaction. Similar studies on purified lens proteins and peptides indicate that this fluorogen is tightly bound to at least one specific peptide. The age related increase in the concentration of fluorescent compounds enhances the yellowing of the lens core and accompanies polymerization of soluble protein precursors into the insoluble protein fraction. There is also a progressive fall in SH levels (with age or UV exposure) and an increase in SS bonds without any significant alteration in the secondary configuration of the lens proteins (which remain mainly in the anti‐parallel beta‐pleated configuration). It is postulated that UV radiation (300–380 nm) plays a role in lenticular aging, particularly nuclear sclerosis and its extreme state characterized by the Brunescent Cataract.

Induction, Acceleration and Prevention (in vitro) of an Aging Parameter in the Ocular Lens

A fluorogen (444-nm emission) is present in mature mammalian ocular lenses (human, rat and mouse) and can serve as an aging parameter in this organ. This fluorogen can be induced in very young human, rat and mouse lenses by incubating them in a media containing 3-aminotriazole and exposing them to ultraviolet (UV) light (300–380 nm). Such in vitro incubations can also accelerate the rate at which this fluorogen normally increases with age; similar results were obtained with in vivo experiments in the mouse. It is proposed that UV-induced free radicals could play a role in this particular aging process. D-Penicillamine is capable of preventing this phenomenon in vitro.

Fluorescence spectra of tryptophan residues in human and bovine lens proteins

Abstract The water soluble proteins from human and bovine lenses have been studied using fluorescence spectroscopy. Excitation of aqueous solutions of α, β and γ-crystallins at 290 nm produced fluorescence emission due solely to tryptophan residues. No emission attributable to tyrosine or phenylalanine residues could be detected. The fluorescence emission maxima for all of the human and bovine crystallins in aqueous solution were in the range 332±2 nm, and the bandwidths were in the range 52±2 nm. These results indicate that the tryptophan residues in these proteins exist mainly in hydrophobic environments in aqueous solution. All of the human and bovine crystallin fractions could be denatured by addition of 8 m -urea, 5 m -guanidine hydrochloride, or 1% sodium dodecyl sulfate (SDS). The former two reagents shifted the fluorescence maxima into the range 349±2 nm, indicating that the majority of tryptophan residues are exposed to water in these solutions. Addition of SDS produced only a small spectral shift to 335±1 nm, but produced a marked spectral broadening to 59±1 nm, again indicating significant protein denaturation. The fluorescence data for lens crystallins have been compared with corresponding data for α-chymotrypsin in an effort to classify the lens crystallin data into discrete spectral classes based on specific fluorescence properties of three types of tryptophan residues.

ULTRAVIOLET ACTION SPECTRUM FOR TRYPTOPHAN DESTRUCTION IN AQUEOUS SOLUTION

The photochemistry of tryptophan plays an important role in numerous photochemical and photobiologkal processes, including enzyme deactivation (Grossweiner et al., 1976; Okumura and Murachi, 1975), and ocular lens protein damage (Borkman et al., 1977; Kurzel et al., 1973). Recent work of McCormick et al. (1976) has demonstrated that hydrogen peroxide is an important product of tryptophan photolysis and that this toxic product is responsible for several of the known biological consequences of tryptophan photolysis. Examples from the older literature may be found in the monograph by McLaren and Shugar

Evidence for a free radical mechanism in aging and u.v.-irradiated ocular lenses

Abstract ESR spectra are reported for whole rat lenses and for the central 3-mm cores from human lenses exposed to u.v. radiation in the ESR cavity at 77°K. The observed signals are attributed to a free radical which is produced via photoionization of tryptophan residues in lens protein. The intensity of the ESR signal was found to be greater in young than in old human lenses and greater in normal lenses than in nuclear (brown) cataracts of the same age. Incubation with penicillamine in the dark reduced the observed ESR signal intensity in rat and human lenses. It is concluded that penicillamine functions as a radical trap within the lens. The ESR experiments reported here, together with evidence from fluorescence spectroscopy, suggest that u.v.-induced damage to the ocular lens could proceed via a free-radical mechanism.

Abinitio studies on the stabilities of even‐ and odd‐membered Hn+ clusters

Ab initio SCF and SD–CI calculations have been performed for the hydrogen positive ion clusters Hn+ with n = 3–9. The optimum structures of the clusters were all determined to consist of a nearly equilateral H3+ core surrounded by neutral H and/or H2 ligands at the apices. The most interesting new finding is that the even‐membered clusters with n = 4, 6, 8 have binding energies comparable to the odd‐membered clusters with n = 5, 7, 9. This result is striking since the odd‐membered clusters have been observed in a number of previous experiments; whereas no even‐membered H+n clusters have ever been observed experimentally. The conclusion appears to be that kinetic (but not thermodynamic) factors preclude formation of H+4, and other even‐membered hydrogen ion clusters, in experiments performed to date. Our best calculations indicate that H4+ is stable by at least 5 kcal/mol relative to H3++H. The other Hn+ species studied have probable binding energies in the range 3–7 kcal/mol.

THE PHOTOLYSIS RATES OF SOME DI- AND TRIPEPTIDES OF TRYPTOPHAN

We have measured the relative rates of photolysis of free tryptophan (trp), the dipeptides Gly‐Trp, Trp‐Gly, Leu‐Trp, and Trp‐Leu, and the tripeptides Gly‐Trp‐Gly and Leu‐Trp‐Leu. The photolyses were performed in neutral 0.1 mM aqueous solutions at 25°C using monochromatic 290 nm Xe arc radiation. Tryptophan loss was monitored by absorption, fluorescence and phosphorescence spectroscopy. The rate of tryptophan fluorescence loss was found to be different in the di‐and tripeptides than in tryptophan monomer. These rate differences depended on both the identity of the neighboring amino acid (gly or leu) and on the nature of the linkage, e.g., the rate of Gly‐Trp photolysis was more than 10 times greater than the rate of Trp‐Gly photolysis. Degassing was found to markedly reduce (factor of 8) the photolysis rates of Trp, Trp‐Gly, and Trp‐Leu, but degassing only slightly reduced (less than a factor of 2) the photolysis rates of the other di‐and tri‐peptides. Photochemical product structures were not determined, but absorption and fluorescence spectra were obtained and products could be inferred in some cases by comparison with data of previous workers. The products appeared to differ greatly among the various peptides studied; Trp, Trp‐Gly, and Trp‐Leu gave oxidation products, while Gly‐Trp and Leu‐Trp apparently gave ring closure products, not requiring oxygen.

Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins.

Fluorescence quenching using acrylamide and iodide quenchers has been used to investigate the microenvironments of tryptophan residues in bovine alpha-, beta-, and gamma-crystallin fractions. Acrylamide quenching is very sensitive to the degree of tryptophan accessibility to the solvent containing the acrylamide. Since acrylamide is able to diffuse into the interior of the protein, accessibility to acrylamide may result from Trp residues lying at the surface of the protein or from the existence of channels leading to the interior of the protein. Iodide ion is hydrated and is limited by its large size and charge to quenching of tryptophan residues lying at or near the surface of proteins. Tryptophan residues in the lens crystallin fractions were found to be highly accessible to acrylamide, yet the rate of quenching by acrylamide was very low, indicating that the tryptophan residues of the lens crystallin fractions occupy predominately hydrophobic environments. The high accessibility to acrylamide likely results from diffusion of acrylamide into the interior of the protein. Accessibility to iodide was much lower, as was the rate of quenching by iodide, adding support to the conclusions from acrylamide quenching.

THE MOLECULAR CHAPERONE FUNCTION OF α-CRYSTALLIN IS IMPAIRED BY UV PHOTOLYSIS

Buffer solutions of the lens protein γ‐crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63°C or to UV radiation at 308 nm. When α‐crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of α‐crystallin. Hence, normal α‐crystallin functioned as a molecular chaperone, providing protection against both UV and heat‐induced protein aggregation. When α‐crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis‐a‐vis both UV and heat‐induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of α‐crystallin was to cause interpeptide crosslinking among the αA2 and αB2 subunits of the α‐crystallin macromolecule. In our experiments α‐crystallin was exposed to UV doses, which resulted in 0, 50 and 90% crosslinking as judged by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. α‐Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated α‐crystallin. The results are consistent with the notion that UV irradiation of α‐crystallin results in loss of chaperone binding sites.