Volume 11, number 3
MOLECULAR • CELLULAR BIOCHEMISTRY
June 15, 1976
I N A C T I V A T I O N O F C A T A L A S E BY N E A R U L T R A V I O L E T LIGHT AND TRYPTOPHAN PHOTOPRODUCTS.* Seymour Z I G M A N , Teresa Y U L O , and Gary A. GRIESS
Division of Ophthalmology, Department of Surgery Ophthalmic Biochemistry Research Laboratory, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642. (Received June 27, 1975)
Summary Certain ocular proteins have been found to be chemically modified by exposure to near-UV light (320-390 nm) in the presence of tryptophan. Colored and fluorescent tryptophan photoproducts bind firmly to proteins, thereby altering their physico-chemical properties. The question of whether such a reaction would inhibit the catalytic action of catalase is herein raised. When solutions of bovine liver catalase were re-incubated up to 24 hr under near-UV with preirradiated tryptophan and dialyzed, most of the ability of the enzyme to decompose H202 was lost. Similar results occurred for catalase activities of bovine cornea and lens epithelia. The enzyme protein exhibited altered UV absorption and fluorescence spectra and increased electrophoretic mobility after binding photoproducts. Near-UV light photoproducts of tryptophan are thus capable of deactivating crystalline and tissue catalase.
Introduction Former work in our laboratory has shown that the eye lens crystallins and aqueous humor serum proteins, are chemically modified by * Supported by a research grant from The National Eye Institute of the National Institutes of Health (EY-00459).
exposure to near-UV light in the presence of tryptophan 1-4. The pigmented and fluorescent photoproducts were firmly bound to amino groups in these proteins, thereby altering their physicochemical properties. The question of whether such a reaction would inhibit important catalytic actions of ocular tissue enzymes is now under consideration, and initial studies of the effects of near-UV light induced tryptophan photoproducts on catalase are herein described. DEISSEROTH and DOUNCE 5 have written an extensive review of the literature concerning catalase structure and function.
Methods and Materials Solutions of L-tryptophan (Eastman Organic Chemicals) at 5 x 10 -3 M in distilled water, p H 7.4 were exposed at 37 °C to an Ultraviolet Products Inc., photochemical grid lamp (PCQ008L; emission principally at 365 nm) for times up to 24 hr at an intensity of 3 mW/cm 2 (measured at 365 nm using a long wave ultraviolet meter, J221, from the same company). Twice crystallized beef liver catalase (Worthington Biochemical Corporation), at a level of 10 -7 M in o.o5 M phosphate buffer, p H 7.5, was incubated at 37 °C under the conditions listed below: (a) in the dark, with no additive (ie: the dark controls); (b) exposed to the U V lamp described above;
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(c) in the dark, but with previously near-UV exposed trytophan added at 5 x 1 0 - 4 M; (d) exposed to the UV lamp with near-UV exposed tryptophan added as above. The reaction mixture was then dialyzed against distilled water for 24 hr with two changes. When ocular tissues were to be assayed, whole calf lenses or whole corneas were incubated in TC 199 medium (Grand Islands Biological Co.) as were the catalase solutions listed above. Assay of catalase activity of lens epithelial cells and corneal epithelial cells was done on their 600 x g (for 10 min) supernatants after homogenization in a Dounce homogenizer containing 20 pooled samples of either tissue in 2 ml of the phosphate buffer. Appropriate dilutions of the 600 × g supernatants were made to allow estimation of catalase activity. Catalase activity was assayed by the spectrophotometric method of BEERS and SIZER6. A solution of enzyme diluted 200,000 fold in water (pH 7.4) was added to a 50 fold dilution of 30% H202 (Merck, Inc.) in phosphate buffer as above, and after thorough mixing, the optical density at 240 nm was measured as a function of time during the first 5 min. Loss of absorption at 240 rim, representing the drop in HzO2 level present, indicated loss of catalase activity. A unit of catalase activity is equal to one micromole of H202 decomposition in one minute. The following methods of chemical and physical observations of the effects of near-UV and tryptophan photoproducts were used. The ultraviolet and visible light absorption spectra of catalase solutions in water were measured in a Cary TM recording spectrophotometer. Binding of photoproducts to catalase was observed by use of 14C-labelled tryptophan as precursor at 50/xC/ml added to 5 x 10-4M tryptophan before U V light exposure, and then by counting isoelectric precipitates of the catalasephotoproduct aggregate in a Beckman Liquid Scintillation Counter with Bray's counting solution. Such precipitates of catalase-Photoproduct aggregates were dark brown. To produce antibodies against catalase, white albino rabbits were injected three times a week for three weeks with i mg of crystalline enzyme in Freund's adjuvant. Double diffusion of antiserum and untreated or treated enzyme at 1 mg/ml was carried out with 2% agarose 150
plates. Amino acid analyses of anzyme hydrolyzed in vacuo for 24 hr in p-toluene sulfonic acid was carried out with a Beckman 120C analyzer. This reagent was used for hydrolysis to prevent oxidation of amino acids during the process 7. For polyacrylamide gel electrophoresis of the treated and untreated enzyme, 5% to 13% gels were used in order to provide data for Ferguson plots. The procedure of ORNSTEIN and DAVIS8 was used with Tris-glycine buffer, p H 8.4, and a current of 4 ma per tube for 1 hr. Staining was with 1% buffalo black in 7% acetic acid, and destaining with 7% acetic acid. Approximately 100 ~g samples were used.
Results Figure 1 illustrates the kinetics of catalase inactivation by its exposure to n-UV light and tryptophan Photo products. Table 1 summarizes the effects of near-UV light and tryptophan photoproducts on the activity of crystalline catalase for 3 and 24 hr exposures. Whereas small losses in H202-decomposing ability were detected for catalase treated with U V only, major losses in this activity occurred with combined photoproduct and U V exposure. The greatest loss of activity was 65 to 70% in the 24 hr combined treatment (ie: photoproducts plus UV-light). As shown in Table 2 lens epithelial cell catalase activity was inhibited in 3 hr by nearly 70% when compared with the 3 hr control. By 24 hr in darkness, lens catalase activity had .800 L
UV-TRY + d V El GHT ~ . ~
'- - " - I
UV-TRY + DARK
5 4 (MIN.)
Fig. 1. Time course of H202 destruction by beef liw:r catalase as influenced by exposure of the crystalline enzyme to near-UV light and/or its photoproducts of tryptophan. (Details of assay are to be found under 'Methods' section).
Table 1 Quantitative details of the influence of near-UV light and/or trytophan photoproducts on the activity of crystalline beef liver catalase in decomposing H202.
Time of tryptophan exposure to near-UV light
3 hours 24 hours
Enzyme exposed to: Dark plus n-UV Near-UV light exposed TRY units* per ml x 104
n-UV light plus n-UV exposed TRY
* One unit of catalase equals one micromole of H202 decomposition per minute• d r o p p e d to 5 0 % of t h e 3hr control. T h e lens c a t a l a s e in n e a r - U V light at 24 h r was f o u n d to b e 3 8 % l o w e r t h a n t h e 24 h r control. Thus, t h e g r e a t e s t loss in lens c a t a l a s e activity d u e to n e a r - U V light a n d t r y p t o p h a n e x p o s u r e t o o k p l a c e d u r i n g t h e first 3 hr. W h e r e a s no loss of c o r n e a e p i t h e l i a l cell c a t a l a s e activity o c c u r r e d in 3 hr, a 5 0 % loss d i d o c c u r in 24 hr as c o m p a r e d with t h e 24 h r control. A s s h o w n in F i g u r e 2, d r a m a t i c c h a n g e s in t h e a b s o r p t i o n s p e c t r a of crystalline c a t a l a s e w e r e o b s e r v e d as a r e s u l t of e x p o s u r e to n e a r - U V light in t h e p r e s e n c e of p h o t o p r o d u c t , b u t slight c h a n g e s w e r e s e e n e v e n in t h e a b s e n c e of light. C o m p l e t e loss of s p e c t r a l d e t a i l a n d l a r g e i n c r e a s e s in o p t i c a l d e n s i t y w e r e o b s e r v e d for e n z y m e e x p o s e d to U V - l i g h t a n d p h o t o p r o d u c t s in t h e r e g i o n of m a x i m u m a b s o r p t i o n of t h e a r o m a t i c a m i n o acids, b u t o p t i c a l d e n s i t y i n c r e a s e s in this r e g i o n w e r e also f o u n d for e n z y m e e x p o s e d to p h o t o p r o d u c t s a l o n e . D e c r e a s e s in t h e S o r e t a b s o r p t i o n at 450 n m a r e b e t t e r i l l u s t r a t e d in F i g u r e 3. N e a r U V light a l o n e d i d n o t a p p r e c i a b l y a l t e r t h e light a b s o r p t i o n profiles of catalase. Table 2 Effects of near-UV light and tryptophan photoproducts on catalase activity in homogenates of epithelial cells of beef cornea and lens.
S o l u t i o n s of c a t a l a s e w e r e i n c u b a t e d a n d e x p o s e d to n e a r - U V o r k e p t in d a r k n e s s for 24 h r as s t a t e d in t h e m e t h o d s section. A f t e r a d d i t i o n of 14C-tryptophan, e i t h e r u n i r r a d i a t e d o r p r e v i o u s l y i r r a d i a t e d for 24 hr, t h e p r o t e i n was i s o e l e c t r i c a l l y p r e c i p i t a t e d . B i n d i n g of p h o t o p r o d u c t to the p r o t e i n was easily o b s e r v e d b e c a u s e the p r e c i p i t a t e o n t h e e x t e n s i v e l y w a s h e d m i l l i p o r e filter was b r o w n . T h e r a d i o activity o n t h e filter p a d of t h e d a r k c o n t r o l ',,,,,,'~ C A T A L A S E + U V - T R Y DARK O9
Tissue Epithelial cells of
in darkness in near-UV light * units per O.D. at 280 nm 3 hr 24 hr 3 hr 24 hr
* One unit of catalase activity is equal to one micromole of H202 decomposition in one minute.
500 400 500 WAVELENGTH
CATAI_ASE + UV-TRY . . . . m, + UV LIGHT
500 400 500 WAVELENGTH
Time of exposure of tryptophan to photoproduct
5 0 0 400 500 WAVELENGTH
300 400 500 WAVELENGTH
Fig. 2. Alterations in the absorption spectrum of beef liver catalase resulting from near-UV light exposure and/or photoproducts of tryptophan. (For details of concentrations and conditions see 'Methods')• 151
OF C A T A L A S E
Fig. 3. Decrease in the 450nm (soret) absorption of crystalline beef liver catalase resulting from exposure to near-UV light and tryptophan photoproducts. The curve labelled dark is for photoproduct added to proteins, but without additional exposure to light.
(containing tryptophan) was 43 cpm, of the near-UV plus tryptophan treated sample, 221 cpm, of the photoproduct-dark sample, 215 cpm, and of the photoproduct-plus nearUV, 324 cpm. Thus preformed photoproduct plus UV exposure caused the greatest binding, a result qualitatively confirmed by the color of the precipitate on the filter pad. However, appreciable binding of the preformed photoproduct also occurred in the dark. Use of unexposed tryptophan plus UV exposure also led to a lesser but definite binding of photoproduct to protein. Extensive dialysis and electrophoresis did not remove the color or radioactivity from these isoelectric precipitates. Another interesting result that can be derived from this experiment is that the isoelectric point required to precipitate the tryptophan-treated proteins ranged from 5.4 to 5.7, whereas that for the photoproduct-treated protein, isoelectric pH's were from 4.25 to 4.5 only. Thus the binding has lowered the isoelectric point, probably by masking positively charged groups, such as amino groups. The negative charge of the photoproducts (electrophoretically determined) also would play a role in lowering the isoelectric point of the protein to which they were bound. The increased electronegativity of the altered catalase as a result of the binding reaction between photoproduct and catalase was also studied by polyacrylamide gel electrophoresis. When the migration distances of the stained band of catalase were measured after the various categories of treatment with near-UV 152
light and/or photoproducts, only the catalase exposed to photoproduct plus UV exposure was found to be more electronegative (21% slower). When studied by use of Ferguson plots, as shown in Figure 4, the free mobility of the dark-maintained sample was 8.7 whereas that of the photoproduct and UV exposed sample was elevated to 13.5 x l0 s cm2/v-s. The retardation coefficients were nearly the same in both samples. This observation indicates a greatly altered charge but little major structural change. The slight increase in retardation could be due to slightly increased hydrophobicity of the protein with bound photoproducts. An altered reaction to anti-catalase serum from rabbits was also found to result from the exposure of catalase to near-UV light in the presence of tryptophan photoproducts. Whereas unincubated catalase, dark-incubated, UVexposed and catalase exposed to photoproducts in the dark all gave unaltered double diffusion precipitin arcs of identity, catalase irradiated with UV-light for 24 hr in the presence of photoproducts of tryptophan showed no precipitin at all. It seems that the antigenic site is BEEF L I V E R C A T A L A S E +
%. 5 o
Fig. 4. Polyacrylamide gel analysis of the effects of near-UV light and tryptophan photoproducts on the electrophoretic mobility of beef liver catalase. Ferguson plots of mobility vs gel concentration are shown.
entirely masked or altered by the bound photoproducts. Amino acid analyses of catalase treated in the above-outlined manner revealed only minor differences in its composition except for the material exposed to UV-light in the presence of photoproducts. The only large changes were in the content of histidine (down by 18%) and in methionine (down 32%), and a great increase in the ammonia level (up by 50%). Smaller decreases of tyrosine, phenylalanine, and tryptophan (estimated by the method of LIN and CHAN6*) were observed, but the values were not down by more than 10%, which is close to the limitation of the method. Similarly, small increases in threonine and serine contents were found, but they were elevated less than 10%. The findings are consistent with the known changes in protein composition resulting from this sensitized photooxidation.
The results show that crystalline bovine liver catalase and the same enzyme as present in calf eye lens and cornea epithelial cells can be inactivated by exposure to near-UV light at intensities not greater than that in sunlight in the presence of tryptophan photoproducts. Whereas exposure of catalase to near-UV light alone or to tryptophan photoproducts in darkness led to a much lesser degree of inactivation, the combined treatment produced nearly a five-fold reduction in catalase activity. The formation of catalase inactivating photoproducts required between 8 and 24 hours of near-UV irradiation of neutral aqueous solutions of tryptophan, and the inactivation of the enzyme required that it be exposed to the photoproducts and the near-UV light for more than 3 hours under the conditions of these experiments. Loss of the catalytic action of catalase appears to result form alterations in some of its chemical and physical properties that are easily observable. The same alterations have already been reported in a purified lens crystallin to result from the binding of photooxidized trytophan to it under similar experimental conditions 4. Thus, the binding of pigmented and fluorescent tryptophan photoproducts (still to be
identified) to the catalase apoprotein led to abnormal light absorption characteristics, increases in electronegativity, destruction of an appreciable number of amino acid residues (ie: histidine and methionine), and an abnormal immunological reactivity. Apparently, some photoproducts bind to amino groups as shown by ZIGMANet al. 4, and/or photosensitize the destruction of other amino acid side chains in or near the active center so as to inactivate the enzyme. This conclusion is supported by the work of NAKATANI9, who reported that a photosensitized loss of catalase light absorption at 405 nm was associated with its inactivation. A question of the physiological significance of the observations reported herein may be raised because of the relatively long periods of UVlight exposure required for photoproduct formation and for catalase inactivation. Low efficiencies of photoproduct production and catalase inactivation must apply. However, the tissues that would be affected by this process would only include the skin and the ocular tissues, which are superficial and in which the turnover of proteins is rather slow. Because the lens epithelial cells are anteriorly situated, and migrate slowly into the interior of the lens where they differentiate and remain indefinitely, periods of exposure to near-UV light could approach or even exceed those used in these experiments. The alterations of proteins would also be cumulative, and since they can continue for over 20 years, may be physiologically significant. Other factors to consider are the presence of photosensitizing substances (ie: riboflavin) and reducing substances in vivo. The function of lens catalase is to break down and relieve the metabolic buildup of H202 in the tissue. Such a buildup of H202 damages lens cells by leading to an increase in oxidation reactions. Increases in oxidizing conditions may be involved in changes in the state of lens proteins leading to protein aggregation and opacities. Cataracts have been described in rabbits resulting from feeding them amizol, a known photosensitizer and catalase inhibitor by BHUYAN and BHUYANTM. While the physiological importance of the experiments reported are still somewhat speculative, inactivation of catalase by tryptophan photoproducts is an example of the possible inactivation of other enzymes, possibly 153
m o r e sensitive t h a n catalase, b y n e a r - U V p h o t o p r o d u c t s , a n d m o r e essential for the livelih o o d of ocular a n d o t h e r tissues.
References 1. Zigman, S., Science 171, 807-809, 1971. 2. Zigman, S., Schultz, J., Yulo, T., and Grover, D., Israel J. Med. Sci. 8, 1590-1595, 1972. 3. Zigman, S., Griess, G., Yulo, T., and Schultz, J., Exp. Eye Res. 15, 255-264, 1973. 4. Zigman, S., Schultz, J., Yulo, T., and Griess, G., Exp. Eye Res. 17,209-217, 1973. 5. Deisseroth, A. and Dounce, A., Physiol Rev. 50, 319-375, 1970. 6. Beers, R. F., and Sizer, I. W., J. Biol. Chem. 195, 133-140, 1952. 7. Ornstein, L. and Davis, B. J., Disc Electrophoresis Distillation Products Industries, Rochester, New York, 1960. 8. Liu, T., and Chang, Y., J. Biol. Chem. 246, 2843-2848, 1971. 9. Nakatani, M., J. Biochem. (Tokyo) 48,476-482, 1960. 10. Bhuyan, K. C. and Bhuyan, D. K., American J. Ophthalmology 69, 147-153, 1973.