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Photooxidation of methionine with immobilized methylene blue photooxidizer
326 Biochimica et Biophysica Acta, 444 (1976) 326--330
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21428 PHOTOOXIDATION OF METHIONINE WITH IMMOBILIZED METHYLENE BLUE AS PHOTOOXIDIZER
CATHERINE LEWIS and WILLIAMH. SCOUTEN Bucknell University, Lewisburg, Pa. 1 7837 (U.S.A.)
(Received June 14th, 1976)
Summary Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84°/0 acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21°/0. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.
Dye-sensitized photooxidations have been used to modify selectively amino acid residues in polypeptides, leading to the clarification of the roles played by those amino acids in protein function. One application of this method, the photooxidation of methionine using free methylene blue as the sensitizer, has been demonstrated by Jori et al. [ 1--3]. In these studies, analysis of the extent of oxidation of methionine was complicated by the presence of the free dye in solution. If the methylene blue is immobilized on glass beads prior to the photooxidation, however, simple gravity filtration removes the catalyst fromcthe reaction mixture. The use of glass beads to immobilize the dye is additionally advantageous in that the beads are stable in a variety of solvents, in acidic or basic media, and they may be stored indefinitely and reused. Dye-sensitized p h o t o o x i d a t i o n using methylene blue is not limited to the oxidation of methionine to methionine sulfoxide. By varying the pH,
327 solvent or temperature, tryptophan, tyrosine, cystine and histidine may be affected as well [3] .Methylene blue is also used in the oxidation of cyclic dienes and polycyclic aromatic c o m p o u n d s to give cyclic peroxides, and of olefins containing allylic hydrogen atoms to give allylic hydroperoxides. A c o m m o n problem in using glass beads to immobilize reactive material is the non-specific adsorption of c o m p o u n d s onto the beads. This report describes a m e t h o d for preparation of the beads which prevents non-specific adsorption while achieving stable coupling between the beads and methylene blue. Porous glass beads, (CPG-550, Corning Biological Products), were obtained from Pierce Chemical Co., Rockford, Ill., U.S.A.; 7-glycidoxypropyltrimethoxysilane (Dow Corning Z-6040 Silane) from Corning Glass Works, Medfield, Mass., U.S.A.; Silica Gel H (type 60) from Merck, Darmstadt, G.F.R.; DL-methionine from Eastman Organic Chemicals, Manchester, N.Y., U.S.A.; lysozyme (Muramidase, chicken egg white, No. L.6876, Lot 74C8040) from Sigma Chemical Co., St. Louis, Mo., U.S.A. Preparation of the methylene blue glass beads was performed as follows. Porous glass beads (1 g) were refluxed gently with ~-glycidoxypropyltrimethoxysilane (5% in 10 ml of toluene) in the presence of p-nitrophenol and sodium p-nitrophenoxide (1°/0 of each in 10 ml of ethanol) for 16 h. After cooling, the beads were filtered by suction and washed with ethanol (60 ml). The aromatic nitro group on the beads was reduced to an aromatic amine by refluxing for 1 h in sodium dithionite (10 ml of a 1% solution in deionized, distilled water), filtered and washed with distilled water (60 ml). Diazotiazation of the amine was performed by adding 2 M HC1 (10 ml), cooling the solution (ice bath, 5°C), and by slowly adding sodium nitrite (250 mg) to the cold solution. Bubbles were removed from the glass pores by evacuating the solution containing the beads for 30 min at 5°C. The beads were filtered and then washed sequentially with cold distilled water, cold aqueous 1% sulfamic acid, and cold distilled water (60 ml of each). The beads were tested at this point for diazonium by reacting a small amount of the beads (10--20 mg) with 1--2 ml of a 1°/0 solution of 2-napthol in 1% NaOH. Beads which have been correctly modified turn red-orange immediately upon addition of the 2-napthol. Methylene blue was coupled to the diazotized glass beads by mixing the beads with a freshly prepared solution of methylene blue (0.05°/0) in aqueous sodium carbonate (20°/0), and containing the reaction in an ice bath with stirring for 1 h. The beads were then filtered and washed sequentially with 100 ml each of distilled water, 950/0 ethanol, chloroform, and ether. Excess methylene blue was further removed by several 5-min refluxes in 10°/0 acetic acid and additional washing. The beads were not used for the photooxidation until the solvents used for washing ceased to remove additional blue color from the beads. Beads prepared in this way are stable for at least a year when stored in 10% acetic acid (Scheme 1). The photooxidation of methionine (10 ml of a 2 mM solution in 500/0 " acetic acid) was carried o u t in test tubes (16 X 150 mm) containing 100--150 mg of the methylene blue beads. T w o controls were run simultaneously: methionine with methylene blue beads, n o t exposed to light (wrapped in
328 OCH 3
J
g ~ ss~ ~- OH
OCH3
OCH3
-Y
OH
ONa
N©2
N©2
J OCH3
i
--~"
OH Na2S2Q5
OCH3 "
L
OCH3
1
OH
NMNO2/HCt
OCH 3
J-
glass))-~O
'I
Si--CH2CId2--OOCH 3
glas~
I
CH2~ CH - - C H 2 -
I
O -
Co>
N2CL
OH
O - - ? i - - - CH2CH2--O - - C H 2 - - ~m - - cm 2 0
~ X / ~
( CH3)2N
N =N,
N (CH3) 2
Scheme1. aluminum foil), and methionine exposed to light but with no beads. Into each tube was placed a Pasteur pipet connected to an O2 tank or to an air source so that 02 or air could be bubbled through the solutions throughout the period of photooxidation. Special care was taken to regulate the flow of 02 or air to achieve complete mixing of the beads and the solutions. The tubes containing the methionine, beads and pipets were placed in a water bath maintained at 37°C, and a plexiglass shield was placed around the entire set up. Four 150 watt tungsten light bulbs were placed outside the shield (2--3 inch from the tubes) and allowed to shine on the reactions for 6 h. Thin-layer chromatography of the reaction mixtures on Silica Gel H in n-butanol/acetic acid/water (4:1:1, v:v:v) and spraying with ninhydrin {0.5o/0 in butanol) showed oxidation of only the sample containing the methionine, methylene blue be~ads and exposed to light. The two control solutions chromatographed with only one spot corresponding to methionine (Rf = 0.38), while the oxidized sample chromatographed with the methionine spot and one additional spot corresponding to methionine sulfoxide (Rf = 0.15). The oxidized sample of methionine was reduced by slowly combining equal volumes of the sample and 1 M 2-mercaptoethanol in 10°/0 sodium
329 carbonate, and adjusting the pH to neutrality with solid Na2COa. The solution was allowed to stand for 1 h. Chromatography as before showed the presence of only one spot which corresponded to methionine. The selective p h o t o o x i d a t i o n of the methionyl residues of lysozyme, which has been shown to be highly sensitive to the pH and to the solvent used [2], was successfully carried out using immobilized methylene blue without adsorption of the enzyme onto the beads. A solution of lysozyme (0.5 mg/ml in 84% acetic acid, 10 ml) was photooxidized according to the procedure described above for methionine for 26 h at 4°C. Two controls were run at the same time: one containing the lysozyme solution, methylene blue beads, but protected from the light, and one containing lysozyme, no beads and exposed to light. Samples of each solution were removed at various intervals so that the course of the reaction could be followed by measuring the decrease in enzyme activity caused by the oxidation of methionine. The activity of the enzyme was measured spectrophotometrically (Beckman DB) at 450 n m by combining 0.2 ml of the photooxidized sample with 2.8 ml of an 0.15 mg/ml solution of Micrococcus lysodeikticus (dried cells, from Sigma Chemical Co., St. Louis, Mo.) in 0.1 M phosphate buffer, pH 7.0. One unit of activity [4] is defined as a decrease in absorbance, at 450 nm, of 0.001 per min at pH 7.0, 25°C. The decrease in per cent specific activity during the course of 26 h of p h o t o o x i d a t i o n for the three solutions in shown in Fig. 1. It can be 100,
c B
LO 3O 2O 10 ;~ 2, 6 8 Ib I~ . . . . . . 26 photooxidation (hi Fig. 1. D e c r e a s e in p e r c e n t a g e specific a c t i v i t y o f l y s o z y m e as a f u n c t i o n of h o u r s o f p h o t o o x i d a t i o n . S o l u t i o n s o f l y s o z y m e ( 0 . 5 m g / m l in 84% a c e t i c a c i d ) w e r e p h o t o o x i d i z e d as d e s c r i b e d in t h e t e x t , a n d a s s a y e d (0.2 m l o f t h e p h o t o o x i d i z e d s a m p l e w i t h 2.8 m l o f a n 0 . 1 5 m g / m l s o l u t i o n o f M i c r o c o c c u s l y s o d e i k t i c u s in 0.1 M p h o s p h a t e b u f f e r , p H 7.0, 2 5 ° C ) for d e c r e a s e in a b s o r b a n c e at 4 5 0 n m o v e r a 4omin i n t e r v a l . One u n i t o f a c t i v i t y is a c h a n g e in a b s o r b a n c e o f 0 . 0 0 1 / r a i n [ 4 ] : specific a c t i v i t y is u n i t s o f a c t i v i t y / r a g e n z y m e . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d [ 5 ] f o r e a c h s a m p l e at e a c h t i m e interval. Line A ( s ) s h o w s t h e p e r c e n t a g e specific a c t i v i t y f o r t h e s a m p l e c o n t a i n i n g l y s o z y m e a n d m e t h y l e n e b l u e - C P G b e a d s a n d e x p o s e d t o light; line B (o) s h o w s t h e p e r c e n t a g e specific a c t i v i t y f o r t h e s a m p l e c o n t a i n i n g l y s o z y m e , n o m e t h y l e n e b l u e - C P G b e a d s a n d e x p o s e d to light; line C (o) s h o w s the p e r c e n t a g e specific a c t i v i t y for t h e s a m p l e c o n t a i n i n g l y s o z y m e a n d m e t h y l e n e b l u e - C P G b e a d s , n o t e x p o s e d t o light. T h e p e r c e n t a g e specific a c t i v i t y w a s o b t a i n e d b y c a l c u l a t i n g t h e r a t i o of t h e specific a c t i v i t y o f e a c h s a m p l e to t h a t of t h e original l y s o z y m e s o l u t i o n t h a t was u s e d f o r t h e r e a c t i o n s . T h e original s o l u t i o n was a s s a y e d f o r a c t i v i t y a n d p r o t e i n c o n t e n t a t t h e s a m e t i m e i n t e r v a l s as t h e samples.
330
seen that the sample containing the lysozyme, methylene blue beads and exposed to light decreased in specific activity by 94%, while the lysozyme solution containing the beads but shielded from the light decreased by only 21°/0, and the solution containing no beads and exposed to light decreased by 33%. As a check for the possible effect of ultraviolet light on the solution containing no beads and exposed to light, the photooxidation was carried out with two modifications. The water bath surrounding the sample tubes during exposure to light was replaced by a bath of cinnamaldehyde which absorbs infinitely between 410 and 200 nm. A third control, containing the lysozyme solution, no beads, and shielded from the light, was added. After 28 h of photooxidation, the added control had the highest remaining specific activity, 66°/0 that of the original activity. The specific activity of the solution containing no beads and exposed to light was still the lowest of the three controls, with 54°/0 specific activity remaining, and the solution containing the beads b u t shielded from light retained 60°/0 of its original activity. Although it can be seen from these data that photooxidation using cinnamaldehyde as a filter caused a greater decrease in the specific activities of all the controls, the importance of these results is that the relative differences between the controls was reduced by the presence of the filter. To show that the decrease in activity of the enzyme was not due to adsorption of the enzyme onto the beads, protein assays [ 5] were performed on each of the samples. If adsorption had occurred, the samples containing the beads would be expected to show a decrease in protein concentration during the period of photooxidation. The results indicate that no adsorption t o o k place. Samples removed from each of the three enzyme solutions at various time intervals throughout the photooxidation period were identical in protein concentration not only to each other but also to the original lysozyme solution that was prepared for the reactions. The authors gratefully acknowledge the assistance, in part, of grants from the Petroleum Research Fund, administered by the American Chemical Society, from the Merck Foundation, from the Research Corporation and from Coming Glass Works. References 1 2 3 4 5
J o r i , G., G a l i a z z o , G. a n d S c o f f o n e , E. ( 1 9 6 9 ) B i o c h e m i s t r y 8, 2 8 6 8 - - 2 8 7 4 . J o r i , G.0 G a l i a z z o , G., M a r z o t t o , A. a n d S e o f f o n e , E. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 4 2 7 2 - - 4 2 7 8 . J o r i , G., G a l i a z z o , G., M a r z o t t o , A. a n d S c o f f o n e , E. ( 1 9 6 8 ) B i o c h i m . B i o p h y s . A c t a 1 5 4 , 1 - - 9 . S h u g a r , D. ( 1 9 5 2 ) B i o c h i m . B i o p h y s . A c t a S, 3 0 2 - - 3 0 9 . L o w r y , O.H., R o s e b r o u g h , N., F a r r , A. a n d R a n d a l l , R. ( 1 9 5 7 ) J. Biol. C h e m . 1 9 3 . 2 6 5 - - 2 7 5 .
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21428 PHOTOOXIDATION OF METHIONINE WITH IMMOBILIZED METHYLENE BLUE AS PHOTOOXIDIZER
CATHERINE LEWIS and WILLIAMH. SCOUTEN Bucknell University, Lewisburg, Pa. 1 7837 (U.S.A.)
(Received June 14th, 1976)
Summary Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84°/0 acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21°/0. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.
Dye-sensitized photooxidations have been used to modify selectively amino acid residues in polypeptides, leading to the clarification of the roles played by those amino acids in protein function. One application of this method, the photooxidation of methionine using free methylene blue as the sensitizer, has been demonstrated by Jori et al. [ 1--3]. In these studies, analysis of the extent of oxidation of methionine was complicated by the presence of the free dye in solution. If the methylene blue is immobilized on glass beads prior to the photooxidation, however, simple gravity filtration removes the catalyst fromcthe reaction mixture. The use of glass beads to immobilize the dye is additionally advantageous in that the beads are stable in a variety of solvents, in acidic or basic media, and they may be stored indefinitely and reused. Dye-sensitized p h o t o o x i d a t i o n using methylene blue is not limited to the oxidation of methionine to methionine sulfoxide. By varying the pH,
327 solvent or temperature, tryptophan, tyrosine, cystine and histidine may be affected as well [3] .Methylene blue is also used in the oxidation of cyclic dienes and polycyclic aromatic c o m p o u n d s to give cyclic peroxides, and of olefins containing allylic hydrogen atoms to give allylic hydroperoxides. A c o m m o n problem in using glass beads to immobilize reactive material is the non-specific adsorption of c o m p o u n d s onto the beads. This report describes a m e t h o d for preparation of the beads which prevents non-specific adsorption while achieving stable coupling between the beads and methylene blue. Porous glass beads, (CPG-550, Corning Biological Products), were obtained from Pierce Chemical Co., Rockford, Ill., U.S.A.; 7-glycidoxypropyltrimethoxysilane (Dow Corning Z-6040 Silane) from Corning Glass Works, Medfield, Mass., U.S.A.; Silica Gel H (type 60) from Merck, Darmstadt, G.F.R.; DL-methionine from Eastman Organic Chemicals, Manchester, N.Y., U.S.A.; lysozyme (Muramidase, chicken egg white, No. L.6876, Lot 74C8040) from Sigma Chemical Co., St. Louis, Mo., U.S.A. Preparation of the methylene blue glass beads was performed as follows. Porous glass beads (1 g) were refluxed gently with ~-glycidoxypropyltrimethoxysilane (5% in 10 ml of toluene) in the presence of p-nitrophenol and sodium p-nitrophenoxide (1°/0 of each in 10 ml of ethanol) for 16 h. After cooling, the beads were filtered by suction and washed with ethanol (60 ml). The aromatic nitro group on the beads was reduced to an aromatic amine by refluxing for 1 h in sodium dithionite (10 ml of a 1% solution in deionized, distilled water), filtered and washed with distilled water (60 ml). Diazotiazation of the amine was performed by adding 2 M HC1 (10 ml), cooling the solution (ice bath, 5°C), and by slowly adding sodium nitrite (250 mg) to the cold solution. Bubbles were removed from the glass pores by evacuating the solution containing the beads for 30 min at 5°C. The beads were filtered and then washed sequentially with cold distilled water, cold aqueous 1% sulfamic acid, and cold distilled water (60 ml of each). The beads were tested at this point for diazonium by reacting a small amount of the beads (10--20 mg) with 1--2 ml of a 1°/0 solution of 2-napthol in 1% NaOH. Beads which have been correctly modified turn red-orange immediately upon addition of the 2-napthol. Methylene blue was coupled to the diazotized glass beads by mixing the beads with a freshly prepared solution of methylene blue (0.05°/0) in aqueous sodium carbonate (20°/0), and containing the reaction in an ice bath with stirring for 1 h. The beads were then filtered and washed sequentially with 100 ml each of distilled water, 950/0 ethanol, chloroform, and ether. Excess methylene blue was further removed by several 5-min refluxes in 10°/0 acetic acid and additional washing. The beads were not used for the photooxidation until the solvents used for washing ceased to remove additional blue color from the beads. Beads prepared in this way are stable for at least a year when stored in 10% acetic acid (Scheme 1). The photooxidation of methionine (10 ml of a 2 mM solution in 500/0 " acetic acid) was carried o u t in test tubes (16 X 150 mm) containing 100--150 mg of the methylene blue beads. T w o controls were run simultaneously: methionine with methylene blue beads, n o t exposed to light (wrapped in
328 OCH 3
J
g ~ ss~ ~- OH
OCH3
OCH3
-Y
OH
ONa
N©2
N©2
J OCH3
i
--~"
OH Na2S2Q5
OCH3 "
L
OCH3
1
OH
NMNO2/HCt
OCH 3
J-
glass))-~O
'I
Si--CH2CId2--OOCH 3
glas~
I
CH2~ CH - - C H 2 -
I
O -
Co>
N2CL
OH
O - - ? i - - - CH2CH2--O - - C H 2 - - ~m - - cm 2 0
~ X / ~
( CH3)2N
N =N,
N (CH3) 2
Scheme1. aluminum foil), and methionine exposed to light but with no beads. Into each tube was placed a Pasteur pipet connected to an O2 tank or to an air source so that 02 or air could be bubbled through the solutions throughout the period of photooxidation. Special care was taken to regulate the flow of 02 or air to achieve complete mixing of the beads and the solutions. The tubes containing the methionine, beads and pipets were placed in a water bath maintained at 37°C, and a plexiglass shield was placed around the entire set up. Four 150 watt tungsten light bulbs were placed outside the shield (2--3 inch from the tubes) and allowed to shine on the reactions for 6 h. Thin-layer chromatography of the reaction mixtures on Silica Gel H in n-butanol/acetic acid/water (4:1:1, v:v:v) and spraying with ninhydrin {0.5o/0 in butanol) showed oxidation of only the sample containing the methionine, methylene blue be~ads and exposed to light. The two control solutions chromatographed with only one spot corresponding to methionine (Rf = 0.38), while the oxidized sample chromatographed with the methionine spot and one additional spot corresponding to methionine sulfoxide (Rf = 0.15). The oxidized sample of methionine was reduced by slowly combining equal volumes of the sample and 1 M 2-mercaptoethanol in 10°/0 sodium
329 carbonate, and adjusting the pH to neutrality with solid Na2COa. The solution was allowed to stand for 1 h. Chromatography as before showed the presence of only one spot which corresponded to methionine. The selective p h o t o o x i d a t i o n of the methionyl residues of lysozyme, which has been shown to be highly sensitive to the pH and to the solvent used [2], was successfully carried out using immobilized methylene blue without adsorption of the enzyme onto the beads. A solution of lysozyme (0.5 mg/ml in 84% acetic acid, 10 ml) was photooxidized according to the procedure described above for methionine for 26 h at 4°C. Two controls were run at the same time: one containing the lysozyme solution, methylene blue beads, but protected from the light, and one containing lysozyme, no beads and exposed to light. Samples of each solution were removed at various intervals so that the course of the reaction could be followed by measuring the decrease in enzyme activity caused by the oxidation of methionine. The activity of the enzyme was measured spectrophotometrically (Beckman DB) at 450 n m by combining 0.2 ml of the photooxidized sample with 2.8 ml of an 0.15 mg/ml solution of Micrococcus lysodeikticus (dried cells, from Sigma Chemical Co., St. Louis, Mo.) in 0.1 M phosphate buffer, pH 7.0. One unit of activity [4] is defined as a decrease in absorbance, at 450 nm, of 0.001 per min at pH 7.0, 25°C. The decrease in per cent specific activity during the course of 26 h of p h o t o o x i d a t i o n for the three solutions in shown in Fig. 1. It can be 100,
c B
LO 3O 2O 10 ;~ 2, 6 8 Ib I~ . . . . . . 26 photooxidation (hi Fig. 1. D e c r e a s e in p e r c e n t a g e specific a c t i v i t y o f l y s o z y m e as a f u n c t i o n of h o u r s o f p h o t o o x i d a t i o n . S o l u t i o n s o f l y s o z y m e ( 0 . 5 m g / m l in 84% a c e t i c a c i d ) w e r e p h o t o o x i d i z e d as d e s c r i b e d in t h e t e x t , a n d a s s a y e d (0.2 m l o f t h e p h o t o o x i d i z e d s a m p l e w i t h 2.8 m l o f a n 0 . 1 5 m g / m l s o l u t i o n o f M i c r o c o c c u s l y s o d e i k t i c u s in 0.1 M p h o s p h a t e b u f f e r , p H 7.0, 2 5 ° C ) for d e c r e a s e in a b s o r b a n c e at 4 5 0 n m o v e r a 4omin i n t e r v a l . One u n i t o f a c t i v i t y is a c h a n g e in a b s o r b a n c e o f 0 . 0 0 1 / r a i n [ 4 ] : specific a c t i v i t y is u n i t s o f a c t i v i t y / r a g e n z y m e . P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d [ 5 ] f o r e a c h s a m p l e at e a c h t i m e interval. Line A ( s ) s h o w s t h e p e r c e n t a g e specific a c t i v i t y f o r t h e s a m p l e c o n t a i n i n g l y s o z y m e a n d m e t h y l e n e b l u e - C P G b e a d s a n d e x p o s e d t o light; line B (o) s h o w s t h e p e r c e n t a g e specific a c t i v i t y f o r t h e s a m p l e c o n t a i n i n g l y s o z y m e , n o m e t h y l e n e b l u e - C P G b e a d s a n d e x p o s e d to light; line C (o) s h o w s the p e r c e n t a g e specific a c t i v i t y for t h e s a m p l e c o n t a i n i n g l y s o z y m e a n d m e t h y l e n e b l u e - C P G b e a d s , n o t e x p o s e d t o light. T h e p e r c e n t a g e specific a c t i v i t y w a s o b t a i n e d b y c a l c u l a t i n g t h e r a t i o of t h e specific a c t i v i t y o f e a c h s a m p l e to t h a t of t h e original l y s o z y m e s o l u t i o n t h a t was u s e d f o r t h e r e a c t i o n s . T h e original s o l u t i o n was a s s a y e d f o r a c t i v i t y a n d p r o t e i n c o n t e n t a t t h e s a m e t i m e i n t e r v a l s as t h e samples.
330
seen that the sample containing the lysozyme, methylene blue beads and exposed to light decreased in specific activity by 94%, while the lysozyme solution containing the beads but shielded from the light decreased by only 21°/0, and the solution containing no beads and exposed to light decreased by 33%. As a check for the possible effect of ultraviolet light on the solution containing no beads and exposed to light, the photooxidation was carried out with two modifications. The water bath surrounding the sample tubes during exposure to light was replaced by a bath of cinnamaldehyde which absorbs infinitely between 410 and 200 nm. A third control, containing the lysozyme solution, no beads, and shielded from the light, was added. After 28 h of photooxidation, the added control had the highest remaining specific activity, 66°/0 that of the original activity. The specific activity of the solution containing no beads and exposed to light was still the lowest of the three controls, with 54°/0 specific activity remaining, and the solution containing the beads b u t shielded from light retained 60°/0 of its original activity. Although it can be seen from these data that photooxidation using cinnamaldehyde as a filter caused a greater decrease in the specific activities of all the controls, the importance of these results is that the relative differences between the controls was reduced by the presence of the filter. To show that the decrease in activity of the enzyme was not due to adsorption of the enzyme onto the beads, protein assays [ 5] were performed on each of the samples. If adsorption had occurred, the samples containing the beads would be expected to show a decrease in protein concentration during the period of photooxidation. The results indicate that no adsorption t o o k place. Samples removed from each of the three enzyme solutions at various time intervals throughout the photooxidation period were identical in protein concentration not only to each other but also to the original lysozyme solution that was prepared for the reactions. The authors gratefully acknowledge the assistance, in part, of grants from the Petroleum Research Fund, administered by the American Chemical Society, from the Merck Foundation, from the Research Corporation and from Coming Glass Works. References 1 2 3 4 5
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