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Reagentless lactate sensor based on cytochrome b2
117 (1980) 115-l 20 Publishing Company, Amsterdam-Printed
Anatytica Chimico Acta, 0 Elsevier
Scientific
REAGENTLESS
J. J. KULYS* Institute (Received
LACTATE
SENSOR
in The
Netherlands
BASED ON CYTOCHROME
b,
and G.-J. S. SVIRMICKAS
of Biocftemistry, 20th November
Lithuanian
Academy
ofScie)lccs.
Viftzios (U.S.S.R.)
1979)
SUMMARY The sensor is based on cytochrome b, adsorbed on the semi-conducting :v-metllylphenazinium-i,i,6,S-tetracganoquinodimethane complex held on a platinum electrode. At an applied potential of -0.03-0.1 V (vs. Ag/AgCI), steady-state currents arc reached in 0.5-0.7 min. The optimal pH is 6.6. Lactate in the range IO-’ --IO-‘ h? can be determined_ The sensor
is useful
for 3-S
days depending
on the enzyme
source.
Two types of enzyme sensors for lactate are known. The first employs cytochrome b, (L-lactate: ferricytochrome c osidoreductase, E. C. 1.1.2.3) to catalyze the osidation of L (+ )-lactate by hesacyanoferrate(II1) [l--3] or by other electron acceptors [4]. The other type is based on the oxidation of L (+)-lactate by nicotinarnide adenosine dinuclcotide (SAD’) catalyzed by lactate dehydrogenase [5, 61. Such lactate sensors based on cytochrotne b2 or on lactate dehydrogenase, with osidation of N_ADH [5] by other compounds, cannot be considered reagentless because of the electron acceptors involved_ Blaedel and Jenkins [6] investigated a reagentless lactate sensor based on N-~-dependent lactate dehydrogenase. However the high anodic potential necessary for the electrochemical osidation of NADH, as well as the short lifetime (less than 30 h), restrict the application of such sensors. The present paper describes a reagentless lactate sensor based on cytochrome bZ adsorbed on a semiconducting charge-transfer complex [7, S] i.e., an organic solid with metal-like electrical conductivity_ EKPERIikIENTAL
Materials The semiconducting compleses used were compleses of I\‘-methylphenazinium (NhlPc) with the anionic radical ‘i,i,S,S-tetracyanoquinodimethane (TCNQ:), NhIP+TCNQ)or NhlPf(TCNQ-)2. They were synthesized by hlalinauskas as described by Melby 191. NhIP (Gee Lawson Chemicals Ltd., England) and TCNQ (Chemapol, Czechosiovakia) were used as received. Cytochrome b, was prepared (at the All Union Research Institute of Applied Enzymology, Vilnius) from Hansenrda anonzala [ 101 with an
activity of 10.473 unit mg -‘- , f_ unit of enzyme activity corresponds to the initial rate of reduction of 1 pmol of hexaeyanoferrate(II1) ;:t pH 7.2. The other solutions used were a 0.1 M phosphate buffer (pH 7.2) prepared from KH2POo and Na,HPOJ, a 1 mkl potassium hexacyanoferrate(If1) solution in the buffer, a IO mM solution of lithium L(f)-lactate (Fermopnost, G.D.R.) in the buffer for the determination of enzyme activity and 39.2-235.2 mM solutions of pure D,L-lactic acid.
of the sensor The finely powdered charge-transfer complex [l-O-1.2 mg) was added, at 2O*C, to 3 ~1 of 0.1 M phosphate buffer (pH 7.2) containing about 0.1 mg of cytochrome b, and mixed thoroughly. The paste obtained was applied to a platinum disk electrode (4.0 mm diameter) and covered with a dialysis membrane (55pm thick). The electrode was stored in the pH 7.2 phosphate buffer solution. Preparation
The sensor current was measured with a polarograph (LP-7e, Czechoslovakia) which had the usual three-electrode circuit. The measurements were carried out in a glass thermostatted cell (10 ml) at 2O+O.l”C. A platinum electrode I(P-101; surface area 56.2 mm’; Radiometer, Denmarkj was used as an ausiliary electrode. A saturated vs. XHE) was used as the reference_
Ag/AgCI
electrode
(+ 0,205 V
The sensor, auxiliary and reference electrodes were first immersed in 8.0 ml of 0.1 X phosphate buffer (pH 7.2) and a suitable potential was applied. The solution was purged with nitrogen for 30 min to remove oxygen. The selected potential, which varied from -0.03 to 0.4 V, was applied until a constant residual current, (i,) was established (this took lo-30 min). Then, 10 1.r1of D,L-kick&e solution was introduced and the change in the anodic current was recorded when the current became constant (0.5-0.7 min). -Another portion of substrate was then added and the steady-state current was again recorded. The difference between the constant currents before and after the lactate additions is proportional to the lactate concentration. RESULTS
In the buffer solution at an anode potential of -0.03 or 0.04 V the residual current of the sensor is 0.01 or 0.02 .uA (Table 1). At more positive potentials the residual current can reach values as high as 0.42 &A. On the introduction of c?,L-lactate the current of the sensor is increased (Fig. 1). The steady-state current is established after 0.5-0.7 min, and remains constant for 15 min or more. Addition of further portions of substrate leads to further current increases. At high concentrations f>3 mM) subsequent increases in the lactate concentration do not change the current (Fig. 2).
117 TABLE Values based
free
1 of
residual
on two
medium;
calculated Anode
and maximal
current
as well
as Michaelis
anode
of lactate
buffer,
sensors
pH 7.2; oxygen-
standard
deviations
of
values 9--12%).
potential
NMP+
NhIP+(TCNQ--‘),
TCNQ:
(V)
;ZA)
‘m&x
K.II(=~~)
ir
‘lnxx
(PAI
(&I)
(NA)
!uA)
0.42
0.40
0.32
3.7
2.1
0.15 0.04 -0.03
0.36 0.02 0.01
3.1 3.0 2.5
2.1 2.1
-
1.9
-
0
constants
complexes (20°C. 0.1 M phosphate potential vs. saturated Ag/AgCl electrode;
charge-transfer
2
r.
6
8
10
Time (mln)
-
5.0
h-xl(app) (mhl)
3.0
-
-
-
-
2 i [C,i_-loctatfzj (mM;
Fig. 1. Changes in the current of the NMPt TCNQ’ sensor with time: (1) 4.90 x lo-‘, (5) 8.35 x lo-;, (6) 1.72 x lo-‘, (2) 1.47 x 10-J. (3) 2.45 x lo-“, (4) 5.40 X lo-‘, (7)2.90X lo-‘,(8)3.44X 10-*,(9)6.39x lo-“,(10)1.23x lo-‘,(11)2.11x lo-’ -0.03 V. hl D.L-lactate. Enzyme 91 unit mg-‘. Applied potential: (a) 0.4 V;(b) Fig. 2. applied
Dependence of the sensor current on potentials (1) 0.4; (2) 0.15; (3) 0.04;
D.L-lactate (4) -0.03
concentration V. Esperimental
at different
conditions
as in Fig. 1.
The current shows a Michaelis dependence on substrate concentration. values obtained from these da’ta agree well, irrespective of the K M(aPP) of the charge-transfer complex anode potential and the composition (Table 1). At lactate concentrations which saturate the electrode response, the higher the anode potential, the higher the maximal current i,, (Table 1). For a sensor based on the 1: 1 complex, i,,, is smaller than that for the 1:2 complex.
In the presence of oxygen (0.25 mM), the sensitivity of thz device in the linear calibration region (0.02-1.2 m&l D,L-lactate) is only 56% ~potenti~ 0.3 V) or 47% (0.08 V) of the sensitivity in anaerobic media. KMtappj is the same in both media, The use of glucose (2.0 r&I) or sucrose (2.0 m&I) instead of lactate does not after the cell current_ In the absence of enzyme the current does not change even at high Ia’ctate concentrations (10 m&I). IYittr increase in the pI-8 from 5.5 to 6.6 (Fig. 3), the current is increased, but the sensitivity decreases above pH 6.6. This compares with the maximum rate of the native enzyme achieved at pH 7.2 (Fig. 3)_ An increase in solution temperature up to 32°C resuits in an increased current (Fig. 4), but higher temperatures lead to lower currents; thereafter, cooling the solution to 20% does not. restore the initial sensitivity. The long-term stability of the sensor at room temperature flS--21°C) was tested; the sensitivity decreased by 45--50% over 3-9 days. The stability of the sensor depends mainly on the sample of cytochrome
b2 used.
DISCUSSION
The action of the sensor is based on the mediator-free e~ectro~hem~~~ of lactate by cytochrome b, [ 111. The hyperbolic dependence of the anodic current on substrate concentration and the bell-shaped pW dependence of the activity, as welt as the temperature dependence indicate oxidation
-.---
-----
- - --’
i
12
Fig. 3. Response of lactate sensor (curve If and native enzyme activity (curve 2) as a fwwtion of pkJ. D.L-LaCt3tC concentration: (1) 0.67 mM, (8) ‘72 mhl; 1.0 mk9 hesatyanofcrrate(If1) (3). Enzyme activity: 10 unit mg-’ (1) and 260 unit mg-’ (2). Other conditions as in Fig. I_
Fig, _s. Change in the sensor current versus temperature for 3.0 mM D.L.-lactate. Enzyme activity, 473 unit mg’ ; applied potential, 0.1 V; aerobic medium at pH 7.2 (0.1 M phosphate buffer).
that the sensor parameters are determined by the enzymatic kinetics. However, diffusion limitations play a considerable role, since the Kllt(app) value of the sensor is an order of magnitude higher than the Michaelis constant for the native enzyme f 12]_ Similar values of KSI(,,,, obtained at different potentials indicate that the value of the potential applied does not influence the efficiency of electron transport by the semiconductor charge-transfer complex. The shift of the optimal pH towards the acidic region, compared with the native enzyme may be accounted for by the change in the local concentration of hydrogen ions on the surface of charge-transfer complexes. According to Goldstein et al. [ 131, the electrostatic potential of organic semiconductor in the region of the enzyme active center is 58.1 mV X A pH, i.e. 34.9 mV, at 20°C. The higher sensitivity of the sensor with increase of temperature is due to the increase in the enzymatic reaction rate, substrate diffusion and the acceleration of the electrochemical process. The change in the enthalpy oi the total process is 10.8 or 6.5 kcal mol- ’ in the temperature ranges 14--22°C or 22-32”C, respectively. Above 32°C. irreversible thermal inactivation of the enzyme takes place, and the sensitivity of the sensor cannot be regenerated. The long-term decrease in sensitivity can be attributed mainly to deactivation of the enzyme. Cytochrome b2 is a complex subunit enzyme [ 141. Its stability is highly dependent on the degree of protease action [lo]. Also, different batches of enzyme used possessed different thermal stabilities. The value of the residual current depends on the anode potential and results from the osidation of the components of the charge-transfer comples f 15]_ Within the range -0.03 to 0.04 V, this current does not esceed 0.6’7% of i max, and thus allows as little as 10 PM L-lactate to be determined. The use of such organic semiconductors and oxidoreductases should allow the
construction
of other mediator-free sensors.
The authors thank Dr. R. Vaitkevieius (The All-Union Research Institute of Applied ~nzymolo~) and Dr. A. Malinauskas for the generous supply of cytochrome b, and the synthesis of organic metals. REFERENCES 1 Ph. Racine, H.-O_ Ktenk and K. Koehsiek. 2. KIin. Chem. Ktin. Biochem., 13 (1975) 533. 2 H. Durliat, M. Comtat, ?. Mahenc and A. Baudras, Anal. Chirn. Acta, 85 (19’76) 31. 3 J_ J. Kulys and G.-J. S, Svirmickas, Anal. Chim. Acta, 109 (1979) 55. 4 J. J. Kulys and G.-J_ S. Svirmickaa, Liet. TSR Mokslu Akad. Darb., Ser. B, 2 (19SO) 9. 5 A. A. Maiinauskaa and J. J- Kulys, Anal. Chim. Acta, 98 (197s) 31. 6 IV_ J. Blaedel and R. A. Jenkins, Anal. Chem., 18 (19’76) 1210. ‘7 E. Engler, Chem. Tech., 274 (1976). 8 J. H. Perlstein, Angew. Chem,, 89 (1957) 534. 9 L. R. Melby, Can. J. Chem., 43 (1965) 1448.
120 10 A. Baudras 11 12 13 1-l
and A. Spyridokis,
Biochimie,
53 (1971)
943.
J. J. Kulys and C.J. S. Svirmickas. Doll. Akad. Nauk SSSR, 245 (1979) 137. J. J. Kulys and K. V. Kadziauskieni, Dokl. Akad. Nauk SSSR, 239 (1978) 636. L. Goldstein, Y. Levin and E. Katchalski, Biochemistry, 3 (1964) 1913. hl. Gervnis, 0. Groudinsky, Y. Risler and F. Labeyrie, Biochem. Biophys.
Commun., 77 (1977) 1543. 15 C. D. Jaeger and A. J. Bard, J. Am.
Chem.
Sot..
101 (1979)
1690.
Res
Anatytica Chimico Acta, 0 Elsevier
Scientific
REAGENTLESS
J. J. KULYS* Institute (Received
LACTATE
SENSOR
in The
Netherlands
BASED ON CYTOCHROME
b,
and G.-J. S. SVIRMICKAS
of Biocftemistry, 20th November
Lithuanian
Academy
ofScie)lccs.
Viftzios (U.S.S.R.)
1979)
SUMMARY The sensor is based on cytochrome b, adsorbed on the semi-conducting :v-metllylphenazinium-i,i,6,S-tetracganoquinodimethane complex held on a platinum electrode. At an applied potential of -0.03-0.1 V (vs. Ag/AgCI), steady-state currents arc reached in 0.5-0.7 min. The optimal pH is 6.6. Lactate in the range IO-’ --IO-‘ h? can be determined_ The sensor
is useful
for 3-S
days depending
on the enzyme
source.
Two types of enzyme sensors for lactate are known. The first employs cytochrome b, (L-lactate: ferricytochrome c osidoreductase, E. C. 1.1.2.3) to catalyze the osidation of L (+ )-lactate by hesacyanoferrate(II1) [l--3] or by other electron acceptors [4]. The other type is based on the oxidation of L (+)-lactate by nicotinarnide adenosine dinuclcotide (SAD’) catalyzed by lactate dehydrogenase [5, 61. Such lactate sensors based on cytochrotne b2 or on lactate dehydrogenase, with osidation of N_ADH [5] by other compounds, cannot be considered reagentless because of the electron acceptors involved_ Blaedel and Jenkins [6] investigated a reagentless lactate sensor based on N-~-dependent lactate dehydrogenase. However the high anodic potential necessary for the electrochemical osidation of NADH, as well as the short lifetime (less than 30 h), restrict the application of such sensors. The present paper describes a reagentless lactate sensor based on cytochrome bZ adsorbed on a semiconducting charge-transfer complex [7, S] i.e., an organic solid with metal-like electrical conductivity_ EKPERIikIENTAL
Materials The semiconducting compleses used were compleses of I\‘-methylphenazinium (NhlPc) with the anionic radical ‘i,i,S,S-tetracyanoquinodimethane (TCNQ:), NhIP+TCNQ)or NhlPf(TCNQ-)2. They were synthesized by hlalinauskas as described by Melby 191. NhIP (Gee Lawson Chemicals Ltd., England) and TCNQ (Chemapol, Czechosiovakia) were used as received. Cytochrome b, was prepared (at the All Union Research Institute of Applied Enzymology, Vilnius) from Hansenrda anonzala [ 101 with an
activity of 10.473 unit mg -‘- , f_ unit of enzyme activity corresponds to the initial rate of reduction of 1 pmol of hexaeyanoferrate(II1) ;:t pH 7.2. The other solutions used were a 0.1 M phosphate buffer (pH 7.2) prepared from KH2POo and Na,HPOJ, a 1 mkl potassium hexacyanoferrate(If1) solution in the buffer, a IO mM solution of lithium L(f)-lactate (Fermopnost, G.D.R.) in the buffer for the determination of enzyme activity and 39.2-235.2 mM solutions of pure D,L-lactic acid.
of the sensor The finely powdered charge-transfer complex [l-O-1.2 mg) was added, at 2O*C, to 3 ~1 of 0.1 M phosphate buffer (pH 7.2) containing about 0.1 mg of cytochrome b, and mixed thoroughly. The paste obtained was applied to a platinum disk electrode (4.0 mm diameter) and covered with a dialysis membrane (55pm thick). The electrode was stored in the pH 7.2 phosphate buffer solution. Preparation
The sensor current was measured with a polarograph (LP-7e, Czechoslovakia) which had the usual three-electrode circuit. The measurements were carried out in a glass thermostatted cell (10 ml) at 2O+O.l”C. A platinum electrode I(P-101; surface area 56.2 mm’; Radiometer, Denmarkj was used as an ausiliary electrode. A saturated vs. XHE) was used as the reference_
Ag/AgCI
electrode
(+ 0,205 V
The sensor, auxiliary and reference electrodes were first immersed in 8.0 ml of 0.1 X phosphate buffer (pH 7.2) and a suitable potential was applied. The solution was purged with nitrogen for 30 min to remove oxygen. The selected potential, which varied from -0.03 to 0.4 V, was applied until a constant residual current, (i,) was established (this took lo-30 min). Then, 10 1.r1of D,L-kick&e solution was introduced and the change in the anodic current was recorded when the current became constant (0.5-0.7 min). -Another portion of substrate was then added and the steady-state current was again recorded. The difference between the constant currents before and after the lactate additions is proportional to the lactate concentration. RESULTS
In the buffer solution at an anode potential of -0.03 or 0.04 V the residual current of the sensor is 0.01 or 0.02 .uA (Table 1). At more positive potentials the residual current can reach values as high as 0.42 &A. On the introduction of c?,L-lactate the current of the sensor is increased (Fig. 1). The steady-state current is established after 0.5-0.7 min, and remains constant for 15 min or more. Addition of further portions of substrate leads to further current increases. At high concentrations f>3 mM) subsequent increases in the lactate concentration do not change the current (Fig. 2).
117 TABLE Values based
free
1 of
residual
on two
medium;
calculated Anode
and maximal
current
as well
as Michaelis
anode
of lactate
buffer,
sensors
pH 7.2; oxygen-
standard
deviations
of
values 9--12%).
potential
NMP+
NhIP+(TCNQ--‘),
TCNQ:
(V)
;ZA)
‘m&x
K.II(=~~)
ir
‘lnxx
(PAI
(&I)
(NA)
!uA)
0.42
0.40
0.32
3.7
2.1
0.15 0.04 -0.03
0.36 0.02 0.01
3.1 3.0 2.5
2.1 2.1
-
1.9
-
0
constants
complexes (20°C. 0.1 M phosphate potential vs. saturated Ag/AgCl electrode;
charge-transfer
2
r.
6
8
10
Time (mln)
-
5.0
h-xl(app) (mhl)
3.0
-
-
-
-
2 i [C,i_-loctatfzj (mM;
Fig. 1. Changes in the current of the NMPt TCNQ’ sensor with time: (1) 4.90 x lo-‘, (5) 8.35 x lo-;, (6) 1.72 x lo-‘, (2) 1.47 x 10-J. (3) 2.45 x lo-“, (4) 5.40 X lo-‘, (7)2.90X lo-‘,(8)3.44X 10-*,(9)6.39x lo-“,(10)1.23x lo-‘,(11)2.11x lo-’ -0.03 V. hl D.L-lactate. Enzyme 91 unit mg-‘. Applied potential: (a) 0.4 V;(b) Fig. 2. applied
Dependence of the sensor current on potentials (1) 0.4; (2) 0.15; (3) 0.04;
D.L-lactate (4) -0.03
concentration V. Esperimental
at different
conditions
as in Fig. 1.
The current shows a Michaelis dependence on substrate concentration. values obtained from these da’ta agree well, irrespective of the K M(aPP) of the charge-transfer complex anode potential and the composition (Table 1). At lactate concentrations which saturate the electrode response, the higher the anode potential, the higher the maximal current i,, (Table 1). For a sensor based on the 1: 1 complex, i,,, is smaller than that for the 1:2 complex.
In the presence of oxygen (0.25 mM), the sensitivity of thz device in the linear calibration region (0.02-1.2 m&l D,L-lactate) is only 56% ~potenti~ 0.3 V) or 47% (0.08 V) of the sensitivity in anaerobic media. KMtappj is the same in both media, The use of glucose (2.0 r&I) or sucrose (2.0 m&I) instead of lactate does not after the cell current_ In the absence of enzyme the current does not change even at high Ia’ctate concentrations (10 m&I). IYittr increase in the pI-8 from 5.5 to 6.6 (Fig. 3), the current is increased, but the sensitivity decreases above pH 6.6. This compares with the maximum rate of the native enzyme achieved at pH 7.2 (Fig. 3)_ An increase in solution temperature up to 32°C resuits in an increased current (Fig. 4), but higher temperatures lead to lower currents; thereafter, cooling the solution to 20% does not. restore the initial sensitivity. The long-term stability of the sensor at room temperature flS--21°C) was tested; the sensitivity decreased by 45--50% over 3-9 days. The stability of the sensor depends mainly on the sample of cytochrome
b2 used.
DISCUSSION
The action of the sensor is based on the mediator-free e~ectro~hem~~~ of lactate by cytochrome b, [ 111. The hyperbolic dependence of the anodic current on substrate concentration and the bell-shaped pW dependence of the activity, as welt as the temperature dependence indicate oxidation
-.---
-----
- - --’
i
12
Fig. 3. Response of lactate sensor (curve If and native enzyme activity (curve 2) as a fwwtion of pkJ. D.L-LaCt3tC concentration: (1) 0.67 mM, (8) ‘72 mhl; 1.0 mk9 hesatyanofcrrate(If1) (3). Enzyme activity: 10 unit mg-’ (1) and 260 unit mg-’ (2). Other conditions as in Fig. I_
Fig, _s. Change in the sensor current versus temperature for 3.0 mM D.L.-lactate. Enzyme activity, 473 unit mg’ ; applied potential, 0.1 V; aerobic medium at pH 7.2 (0.1 M phosphate buffer).
that the sensor parameters are determined by the enzymatic kinetics. However, diffusion limitations play a considerable role, since the Kllt(app) value of the sensor is an order of magnitude higher than the Michaelis constant for the native enzyme f 12]_ Similar values of KSI(,,,, obtained at different potentials indicate that the value of the potential applied does not influence the efficiency of electron transport by the semiconductor charge-transfer complex. The shift of the optimal pH towards the acidic region, compared with the native enzyme may be accounted for by the change in the local concentration of hydrogen ions on the surface of charge-transfer complexes. According to Goldstein et al. [ 131, the electrostatic potential of organic semiconductor in the region of the enzyme active center is 58.1 mV X A pH, i.e. 34.9 mV, at 20°C. The higher sensitivity of the sensor with increase of temperature is due to the increase in the enzymatic reaction rate, substrate diffusion and the acceleration of the electrochemical process. The change in the enthalpy oi the total process is 10.8 or 6.5 kcal mol- ’ in the temperature ranges 14--22°C or 22-32”C, respectively. Above 32°C. irreversible thermal inactivation of the enzyme takes place, and the sensitivity of the sensor cannot be regenerated. The long-term decrease in sensitivity can be attributed mainly to deactivation of the enzyme. Cytochrome b2 is a complex subunit enzyme [ 141. Its stability is highly dependent on the degree of protease action [lo]. Also, different batches of enzyme used possessed different thermal stabilities. The value of the residual current depends on the anode potential and results from the osidation of the components of the charge-transfer comples f 15]_ Within the range -0.03 to 0.04 V, this current does not esceed 0.6’7% of i max, and thus allows as little as 10 PM L-lactate to be determined. The use of such organic semiconductors and oxidoreductases should allow the
construction
of other mediator-free sensors.
The authors thank Dr. R. Vaitkevieius (The All-Union Research Institute of Applied ~nzymolo~) and Dr. A. Malinauskas for the generous supply of cytochrome b, and the synthesis of organic metals. REFERENCES 1 Ph. Racine, H.-O_ Ktenk and K. Koehsiek. 2. KIin. Chem. Ktin. Biochem., 13 (1975) 533. 2 H. Durliat, M. Comtat, ?. Mahenc and A. Baudras, Anal. Chirn. Acta, 85 (19’76) 31. 3 J_ J. Kulys and G.-J. S, Svirmickas, Anal. Chim. Acta, 109 (1979) 55. 4 J. J. Kulys and G.-J_ S. Svirmickaa, Liet. TSR Mokslu Akad. Darb., Ser. B, 2 (19SO) 9. 5 A. A. Maiinauskaa and J. J- Kulys, Anal. Chim. Acta, 98 (197s) 31. 6 IV_ J. Blaedel and R. A. Jenkins, Anal. Chem., 18 (19’76) 1210. ‘7 E. Engler, Chem. Tech., 274 (1976). 8 J. H. Perlstein, Angew. Chem,, 89 (1957) 534. 9 L. R. Melby, Can. J. Chem., 43 (1965) 1448.
120 10 A. Baudras 11 12 13 1-l
and A. Spyridokis,
Biochimie,
53 (1971)
943.
J. J. Kulys and C.J. S. Svirmickas. Doll. Akad. Nauk SSSR, 245 (1979) 137. J. J. Kulys and K. V. Kadziauskieni, Dokl. Akad. Nauk SSSR, 239 (1978) 636. L. Goldstein, Y. Levin and E. Katchalski, Biochemistry, 3 (1964) 1913. hl. Gervnis, 0. Groudinsky, Y. Risler and F. Labeyrie, Biochem. Biophys.
Commun., 77 (1977) 1543. 15 C. D. Jaeger and A. J. Bard, J. Am.
Chem.
Sot..
101 (1979)
1690.
Res