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Detection of complex of d-amino-acid oxidase and d-alanine by rapid scan spectrophotometry
I84
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 63338
Detection of complex of D-amino-acid oxidase and D-alanine by rapid scan spectrophotometry The existence of complexes of t)-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, pyruvate or phenylpyruvate etc. was proved spectrophotometrically by the titration of the enzyme with these materials r 4. The characteristic change of the absorption si)ectrum of the enzyme during the titration is seen in a sharpening of peaks at the 42o, 45o and 49o m# absorption bands as well as, in some cases, a red shift of these peaks. It was KUBO AND SmGA z who first observed a complex formation of D-amino-acid oxidase and D-alanine. They measured spectral changes with time at various wavelengths and recorded the spectrum of the complex at a fixed time. MASSEY AN1) GIBSON':'used the same method to obtain kinetic constants of the reaction, but they did not determine the spectrum of the initial complex. In order to deternfine the precise spectrum of the short lived initial complex, a new approach has been desired. Though the time scan method is advantageous for the kinetic analysis and the deternfination of the spectra of longer lived intermediates, this method seems unreliable for our present purpose. Later YAGI, OZAWAAND NAOZGreported a complex of the enzyme and D-lactate. Since the reduction of enzyme with D-lactate was much slower than that with I)-alanine, the measurement of spectral change was easy. However, when we take into consideration its extremely low reactivity, D-lactate did not seem to be a proper substrate of the enzynle.
The purpose of this communication is to report the complex formation of D-amino-acid oxidase and D-alanine, as observed by a rapid scan spectrophotometer, equipped with an analogue data recorder used for external memory. The benzoate-free D-amino-acid oxidase was prepared as described previously 7-9. The D-alanine used was the product of General Biochemicals. A Hitachi RPS-2 rapid scan spectrophotometer was used for measuring absorption spectra. The scan time of wavelength from 42o to 52o m# is 15o msee, and its frequency is 3 cycles/see. Signals of the absorption against time, A - - A ( t ) , and that of wave.length, 3. : ;t(t), were recorded sinmltaneously in each channel of the TEA(; 35I-F data recorder. After the reaction, some of the stored spectra were selected by a sweep controller and displayed on Hitachi V-oI8 memoriscope, reducing the tape speed to one fourth of the recording speed.
'
Rapid s c a r
I
~--~I
~F~-~ a oqu~, dato
F .....
[---'~
~4
Sweep
p .....
[Memor:scope -4w or
I i
:spectrometerF ..4 (5~ r$c(orderLA' =A(nt) J controller~Ak =Ak (nt)qX-Y recorderI'. ),=X(t)
%k =
%k
(nt)
Fig. z. Block diagram of s p e c t r o p h o t o m c t c r used. Solid lint" indicates recording of signals of absorption with time, .4 - A (t), and wavelength, ). ~(t). The dotted line shows the reproduction of A -- .4 (nt) and ). = ).(n!), where n indicates the rate of reduction speed. Desired a b s o r p t i o n spectra were selected by tile sweep controller and displayed on the memoriscope. Biochim. Biohhys. Acla,
I()7 (I968)
z84-I86
SHORT COMMUNICATIONS
I8 5
Fig. I shows the block diagram of our equipment. The analogue recorder, serving as an external memory, allowed us to follow and store all informations of rapidly changing spectra, and to reproduce them repeatedly by reducing the speed of display. This method has a draw-back that it is an off-line system in data processing, trot nevertheless it made it much easier to detect the absorption spectrum of a short life coinplex. Also it has become unnecessary to reduce the rate of enzymatic reaction using abnormal experimental conditions. The result is shown in Fig. 2. After aerobic addition of t~-alanine to D-aminoacid oxidase, an immediate spectral change was observed. The spectrum is quite similar to that of other complexes of D-amino-acid oxidase. While oxygen in the solution was being consumed, the spectrum remained unchanged. Later, however, after the anaerobiosis (3o sec) a bleaching of flavin started to make, first, a purple form, and then a reduced forin of the enzyme ~°. There remained some possibility that this spectrum did not show the complex of an enzyme substrate, but that of an enzyme-product. As pyruvate was formed during oxygen consumption, this spectral change may be considered due to enzyme-
±
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J
520
470
420
520
470
420
5 0
0.1A -¢-
.£ -f
0.1A
J
4 0
420
m~
F i g . z. C h a n g e o f t h e a b s o r p t i o n s p e c t r u m o f D - a m i n o - a c i d o x i d a s e ( 6 . t o ~ M) b y a e r o b i c a d d i t i o n o f o - a l a n i n e (2.5 " t o -3 M) a t 15 '~ ( p H 7.o). A. B e f o r e a d d i t i o n o f s u b s t r a t e . B. 1.2 sec a f t e r a d d i t i o n . C. 37 sec. D. 42 sec. E . . I 5 sec. F. 49 sec. B y t h e m e a s u r e m e n t o f o x y g e n c o n s u m p t i o n in t h e s a m e e x p e r i m e n t a l c o n d i t i o n , a n a e r o b i o s i s w a s c o n f i r m e d t o b e a t t a i n e d a t 3 ° sec a f t e r s t a r t i n g of the reaction.
pyruvate complex. However, the dissociation constant of enzyme and pyruvate was estimated as about IO 2 M, whereas oxygen content in ttle solution was 2 - I O * M and flavin of the enzyme 6" IO-5 M. Therefore, the possible amount of produced pyruvate was calculated as 2.6-IO-4 M (ref. I). When 5" IO-4 M pyruvate was added aerobically to the enzyme, no spectral change could be observed. Tile complex between the oxidized enzyme and imino acid was also considered s. According to tile scheme of MASSEY ANt) GIBSON'*, in anaerobic condition there is no possibility of the existence Biochim. Biophys. Acta, x67 ( t 9 6 8 ) 1 8 4 - 1 8 0
186
SHORT COMMUNICATIONS
of oxidized enzyme-imino acid complex..,ks shown in Fig. 2 (C and D), the shoulder peak of absorption at 49 ° m# could be still observed, after the oxygen in solution was exhausted as indicated by the measurement of oxygen consumption. Therefore, the I)ossibility of the existence of the above complex seems to be negative. "ftms the complex should be considered to be that of the enzyme and substrate. This work was supported by a grant from the Japanese Ministry of Education for scientific research.
Department of Physicochemical Physiology, and Biomedical Research Center*, Medical School, Osaka University, Osaka (Japan)
r 2 3 4 5 6
7 ~, ~J lo
HIROSH! WATARI AKIO IsorvlOTO HAJIME ODA M. KURODA*
11. K u B o AND T. SItIGA, l~ull. Soc. Chin*. Biol., 44 (I9 (~2) 057. I'(. YAGI ANr~ T. Q)ZAX.t,'A, 13zochim. Biophys. ,.tcta, 50 (I962) 42o. V. Masm.:v ,xYr~ H. GANTm'R, Biochemistry, .5 (z965) 1161. K..XSHn)A, J. Biochem. Tokyo, ~l (1`4(~7) 433. V. I~IAS~,I.;Y At;I) (7. I1. GZBSON, Federation Pro&, 23 (t904) 18. K. YAGt, T. OZAWA AXl) M. NAOL J. Biochem. "l'okyo, 5(~ (1964) 487 • H. V,l:~o, T. Ya.xIA.~o, M. IWATSV~O, H. \VATARL T. SHmA AND A. l s o x m a ' o , Bull. Soc. Chim. Biol., 42 (19(~o) 569. V. 31ASSEY ANI) (..;. PALMI'~R, Iliochem. J., 74 (~96o) .to. K. MAGI AND '1". ()ZA%'A, Biochim. lhophy~. ,.'lcta, .5(~ (t962) 413 • H. l,~uno, H. \.~:ATARI AND Z. SItIGA, Bull. Soc. (;him. t3~ol., 41 (1Q59) ()NI.
Received April 22nd, I9()8 Biochim. Bioph.vs. Acta, 1(,7 (19(~8) t,~4-1815
~BA 63335
Betaine aldehyde dehydrogenase: assay and partial purification Betaine aldehyde dehydrogenase (betaine-aldehyde:NAD oxidoreductase, EC 1.2.1.8) is the enzyme responsible for the oxidation of betaine aldehyde to betaine. The activity of this enzyme has been previously measured either manometrically a or spectrophotometrically". The present comnmnication describes a procedure for the partial purification and the necessary conditions for the fluorometric assay of the enzyme. Assay. The buffer substrate was prepared just prior to each analysis and contained the following constituents at the indicated final concentrations: IOO mM Tris buffer (pH 8.1) ; 5 mM Cleland's reagent (Calbiochem.) ; I mM NAD+; and 4 mM betaine aldehyde. The betaine aldehyde was prepared from 2,2-diethoxy-ethyltrimethylammonium iodide (Aldrich, D837o) as described by ROTHSCHILD Agl) GUZMAN-BA~RONa. The stock solution was approx. 4oo mM and was stored at - 2 o °. I M1of water or enzynTe (equivalent to o.4#g protein) was added to a pointed tube of 2.5 mm internal diameter containing IO/~1 of ice-cold incubation mixture. The Biochim. l~iophys. Acta, t67 (I,468) 186-189
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 63338
Detection of complex of D-amino-acid oxidase and D-alanine by rapid scan spectrophotometry The existence of complexes of t)-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, pyruvate or phenylpyruvate etc. was proved spectrophotometrically by the titration of the enzyme with these materials r 4. The characteristic change of the absorption si)ectrum of the enzyme during the titration is seen in a sharpening of peaks at the 42o, 45o and 49o m# absorption bands as well as, in some cases, a red shift of these peaks. It was KUBO AND SmGA z who first observed a complex formation of D-amino-acid oxidase and D-alanine. They measured spectral changes with time at various wavelengths and recorded the spectrum of the complex at a fixed time. MASSEY AN1) GIBSON':'used the same method to obtain kinetic constants of the reaction, but they did not determine the spectrum of the initial complex. In order to deternfine the precise spectrum of the short lived initial complex, a new approach has been desired. Though the time scan method is advantageous for the kinetic analysis and the deternfination of the spectra of longer lived intermediates, this method seems unreliable for our present purpose. Later YAGI, OZAWAAND NAOZGreported a complex of the enzyme and D-lactate. Since the reduction of enzyme with D-lactate was much slower than that with I)-alanine, the measurement of spectral change was easy. However, when we take into consideration its extremely low reactivity, D-lactate did not seem to be a proper substrate of the enzynle.
The purpose of this communication is to report the complex formation of D-amino-acid oxidase and D-alanine, as observed by a rapid scan spectrophotometer, equipped with an analogue data recorder used for external memory. The benzoate-free D-amino-acid oxidase was prepared as described previously 7-9. The D-alanine used was the product of General Biochemicals. A Hitachi RPS-2 rapid scan spectrophotometer was used for measuring absorption spectra. The scan time of wavelength from 42o to 52o m# is 15o msee, and its frequency is 3 cycles/see. Signals of the absorption against time, A - - A ( t ) , and that of wave.length, 3. : ;t(t), were recorded sinmltaneously in each channel of the TEA(; 35I-F data recorder. After the reaction, some of the stored spectra were selected by a sweep controller and displayed on Hitachi V-oI8 memoriscope, reducing the tape speed to one fourth of the recording speed.
'
Rapid s c a r
I
~--~I
~F~-~ a oqu~, dato
F .....
[---'~
~4
Sweep
p .....
[Memor:scope -4w or
I i
:spectrometerF ..4 (5~ r$c(orderLA' =A(nt) J controller~Ak =Ak (nt)qX-Y recorderI'. ),=X(t)
%k =
%k
(nt)
Fig. z. Block diagram of s p e c t r o p h o t o m c t c r used. Solid lint" indicates recording of signals of absorption with time, .4 - A (t), and wavelength, ). ~(t). The dotted line shows the reproduction of A -- .4 (nt) and ). = ).(n!), where n indicates the rate of reduction speed. Desired a b s o r p t i o n spectra were selected by tile sweep controller and displayed on the memoriscope. Biochim. Biohhys. Acla,
I()7 (I968)
z84-I86
SHORT COMMUNICATIONS
I8 5
Fig. I shows the block diagram of our equipment. The analogue recorder, serving as an external memory, allowed us to follow and store all informations of rapidly changing spectra, and to reproduce them repeatedly by reducing the speed of display. This method has a draw-back that it is an off-line system in data processing, trot nevertheless it made it much easier to detect the absorption spectrum of a short life coinplex. Also it has become unnecessary to reduce the rate of enzymatic reaction using abnormal experimental conditions. The result is shown in Fig. 2. After aerobic addition of t~-alanine to D-aminoacid oxidase, an immediate spectral change was observed. The spectrum is quite similar to that of other complexes of D-amino-acid oxidase. While oxygen in the solution was being consumed, the spectrum remained unchanged. Later, however, after the anaerobiosis (3o sec) a bleaching of flavin started to make, first, a purple form, and then a reduced forin of the enzyme ~°. There remained some possibility that this spectrum did not show the complex of an enzyme substrate, but that of an enzyme-product. As pyruvate was formed during oxygen consumption, this spectral change may be considered due to enzyme-
±
/ f
J
520
470
420
520
470
420
5 0
0.1A -¢-
.£ -f
0.1A
J
4 0
420
m~
F i g . z. C h a n g e o f t h e a b s o r p t i o n s p e c t r u m o f D - a m i n o - a c i d o x i d a s e ( 6 . t o ~ M) b y a e r o b i c a d d i t i o n o f o - a l a n i n e (2.5 " t o -3 M) a t 15 '~ ( p H 7.o). A. B e f o r e a d d i t i o n o f s u b s t r a t e . B. 1.2 sec a f t e r a d d i t i o n . C. 37 sec. D. 42 sec. E . . I 5 sec. F. 49 sec. B y t h e m e a s u r e m e n t o f o x y g e n c o n s u m p t i o n in t h e s a m e e x p e r i m e n t a l c o n d i t i o n , a n a e r o b i o s i s w a s c o n f i r m e d t o b e a t t a i n e d a t 3 ° sec a f t e r s t a r t i n g of the reaction.
pyruvate complex. However, the dissociation constant of enzyme and pyruvate was estimated as about IO 2 M, whereas oxygen content in ttle solution was 2 - I O * M and flavin of the enzyme 6" IO-5 M. Therefore, the possible amount of produced pyruvate was calculated as 2.6-IO-4 M (ref. I). When 5" IO-4 M pyruvate was added aerobically to the enzyme, no spectral change could be observed. Tile complex between the oxidized enzyme and imino acid was also considered s. According to tile scheme of MASSEY ANt) GIBSON'*, in anaerobic condition there is no possibility of the existence Biochim. Biophys. Acta, x67 ( t 9 6 8 ) 1 8 4 - 1 8 0
186
SHORT COMMUNICATIONS
of oxidized enzyme-imino acid complex..,ks shown in Fig. 2 (C and D), the shoulder peak of absorption at 49 ° m# could be still observed, after the oxygen in solution was exhausted as indicated by the measurement of oxygen consumption. Therefore, the I)ossibility of the existence of the above complex seems to be negative. "ftms the complex should be considered to be that of the enzyme and substrate. This work was supported by a grant from the Japanese Ministry of Education for scientific research.
Department of Physicochemical Physiology, and Biomedical Research Center*, Medical School, Osaka University, Osaka (Japan)
r 2 3 4 5 6
7 ~, ~J lo
HIROSH! WATARI AKIO IsorvlOTO HAJIME ODA M. KURODA*
11. K u B o AND T. SItIGA, l~ull. Soc. Chin*. Biol., 44 (I9 (~2) 057. I'(. YAGI ANr~ T. Q)ZAX.t,'A, 13zochim. Biophys. ,.tcta, 50 (I962) 42o. V. Masm.:v ,xYr~ H. GANTm'R, Biochemistry, .5 (z965) 1161. K..XSHn)A, J. Biochem. Tokyo, ~l (1`4(~7) 433. V. I~IAS~,I.;Y At;I) (7. I1. GZBSON, Federation Pro&, 23 (t904) 18. K. YAGt, T. OZAWA AXl) M. NAOL J. Biochem. "l'okyo, 5(~ (1964) 487 • H. V,l:~o, T. Ya.xIA.~o, M. IWATSV~O, H. \VATARL T. SHmA AND A. l s o x m a ' o , Bull. Soc. Chim. Biol., 42 (19(~o) 569. V. 31ASSEY ANI) (..;. PALMI'~R, Iliochem. J., 74 (~96o) .to. K. MAGI AND '1". ()ZA%'A, Biochim. lhophy~. ,.'lcta, .5(~ (t962) 413 • H. l,~uno, H. \.~:ATARI AND Z. SItIGA, Bull. Soc. (;him. t3~ol., 41 (1Q59) ()NI.
Received April 22nd, I9()8 Biochim. Bioph.vs. Acta, 1(,7 (19(~8) t,~4-1815
~BA 63335
Betaine aldehyde dehydrogenase: assay and partial purification Betaine aldehyde dehydrogenase (betaine-aldehyde:NAD oxidoreductase, EC 1.2.1.8) is the enzyme responsible for the oxidation of betaine aldehyde to betaine. The activity of this enzyme has been previously measured either manometrically a or spectrophotometrically". The present comnmnication describes a procedure for the partial purification and the necessary conditions for the fluorometric assay of the enzyme. Assay. The buffer substrate was prepared just prior to each analysis and contained the following constituents at the indicated final concentrations: IOO mM Tris buffer (pH 8.1) ; 5 mM Cleland's reagent (Calbiochem.) ; I mM NAD+; and 4 mM betaine aldehyde. The betaine aldehyde was prepared from 2,2-diethoxy-ethyltrimethylammonium iodide (Aldrich, D837o) as described by ROTHSCHILD Agl) GUZMAN-BA~RONa. The stock solution was approx. 4oo mM and was stored at - 2 o °. I M1of water or enzynTe (equivalent to o.4#g protein) was added to a pointed tube of 2.5 mm internal diameter containing IO/~1 of ice-cold incubation mixture. The Biochim. l~iophys. Acta, t67 (I,468) 186-189