- Email: [email protected]
Reversal in the kinetics of the M state decay of D96N bacteriorhodopsin: probing of enzyme catalyzed reactions
B
ELSEVIER
Sensorsand ActuatorsB 35-36 (1996) 470--474
eHe-fi'~V~Zt
Reversal in the kinetics of the M state decay of D96N bacteriorhodopsin: probing of enzyme catalyzed reactions P.C. P a n d e y a,*, S u d h a S i n g h a, B i p i n U p a d h y a y a, H o w a r d H. W e e t a U b, P e t e r K. C h e n c aDepartmen¢of Chemistry, BanarasHindu University, Varanasi221005, India bBiotechnology Division. NationalInstituteof Standards and Technology. Gaithersburg. MD 20899. USA cDelmrfmentof Biology, GeorgetownUniversity, Washington,DC 20057, USA
Abstract The kinetics of M state decay of the D96N bacteriorhodopsin (BR) in the presence of (i) ammonium ion and (ii) an aromatic amine is reported. The M state decay rate becomes faster in the presence of ammonium salt with a concomitant decrease in absorbance maxima at 410 nm. in the presence of an aromatic amine, a slower M state decay rate with an increase in absorbance maxima at 410 nm is observed. The variation in the kinetics of M-state decay is due to the change in the lifetime of the M-state associated with the conversion of M-intermediate to trans-BR. The decay rate of the M-state has also been found to be greatly affected in the presence of enzymatic reactions resulting in the formation of either an acid or a base. Three enzymatic reaction systems, urease, acetylcholinesterase and penicillinase, have been used to study the kinetics of M state decay of D96N bacteriorhodopsin. The kinetic data are found to be dependent on the concentrations of the respective enzyme's substrate. Results of the studies using urea, acetylcholine and penicillin are reported. The enzymes were also immobilized together with D96N bacteriorhodopsin in sol-gel glass to develop a biosensor for the analysis of urea, acetylcholine and penicillin. Keywords: M state decay; Kinetics; D96N bacteriorhodopsin
1, Intredection Over the last few years [1-22] there has been increased attention focused on the study of the photoactivity of bacteriorhodopsin (BR). The purple membrane protein of Haiobacterium solinarium (previously Halobacterium halobium) acts as a proton-pump after photoexcitation, transporting protons from the cytoplasmic side of the membrane to the extracellular environment against a concentration gradient [10,23]. The photocycle consists of a series of steps which are initiated on absorption of a photon. The kinetics of photoexcitation ultimately determines the behavior of proton-pumping and light absorption efficiency of the bacteriorhodopsin (BR). The proton pumping results in a pH gradient based on directed sequential changes of the pKa of the retinal Schiff base [11.24]. During the photocycle, the proton translocation is accomplished by the deprotonation of a Schiff base followed by * Conespondingauthor.
ElsevierSdenceS.A. PII
S0925-4005(96)02083-7
reprotonation. The deprotonation step produces the M state intermediate from the K and L intermediates which are formed subsequently once retinal is isomerized in the presence of an external light source. The reprotonation initiates the M state decay in the absence of an external light source. There are several reports [7,8,11] describing proton uptake in bacteriorhodopsin. Retinal isomerization reactions are important in the proton pumping cycle of bacteriorhodopsin [10]. The retinal undergoes an all-trans 13-cis or an all-trans-~ 13,14-di-cis photoisomerization as a very fast primary step in the photocycle forming the K intermediate. The initial retinal isomerization step, although very fast (3 ps), has been found to be dependent on internal water molecules [10]. The water affects the nature of the Schiff base counter ion and the nature of the primary photoreaction. It was suggested that water chains subsequently effect the reprotonation and deprotonation steps of the proton pump photocycle [10]. The deprotonation of L state (an intermediate formed from the K in-
P.C. Pandey et al. / Sensors and Actuators 1735-36 (1996) 470--474
termediate) leads to the formation of M state which decays back to trans-BR in the absence of an external light source. The presence of the M state can be monitored by the measurement of the absorption at 410 nm. It has been observed that the lifetime of the Mintermediate in the BR photocycle was remarkably prolonged on drying a suspension of purple membrane [25] in the presence of arginine. Guanidine has also been reported to increase the lifetime of M state of BR in a similar fashion [26]. It is important to understand the effects of the proton donor/acceptor present on the extracellular side with respect to the lifetime of the M state. The lifetime of the M state eventually determines decay rate back to the ground state (trans-BR) after reisomerization in the dark which is a slower step of the photocycle. We chose to use ammonium ion and a primary aromatic amine to modify the kinetics of M state decay since these molecules may effect the lifetime of the M state in the photocycle. The effect of pH on the retinal reisomerization has been reported by several workers [7,8,13,141, At low pH, the retinal reisomerization occurs first, whereas at high pH, this is delayed. The delay in retinal reisomerization results in a slower decay of the M state. The pH dependence of retinal reisomerization directed our attention to study the decay rate of the M state in the presence of the enzymatic systems leading to the formation of either an acid or a base. This type of reaction will affect the kinetics of M state decay of the D96N mutant BR. This change in the kinetics of the M state decay can thus be used in monitoring the enzymatic reactions associated with the formation of either an acid or a base. This relationship may he useful in the development of future BR containing analytical devices. In this payer we report the kinetics of the M state decay of DetiN BR in the presence of ammonium salt and benzylamine. The enzymatic reactions employing urease, acetylcholinesterase and peniciUinase has also been monitored, based on the measurement of the kinetics of the M state decay in the dark. These enzymes together with bacteriorhodopsin have been immobilized in a solgel glass as a first step in the development of a biosensor for the analysis of urea, acetyicholine and penicillin.
2. Experimental 2.1. Materials The materials used in this investigation and their sources were as follows: The D96N mutant bacteriorhodopsin used in this investigation was supplied by Dr A.B. Druzhko0 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchina, Russia. Urease, acetylcholinesterase, penicillinase, benzylamine, penicillin G, acetylcho|ine were obtained form Sigma Chemical Company (St. Louis, MO); Tetrameth-
471
oxy-silane (TMOS) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). All other chemicals used were of analytical grade.
2.2. Measurement of absorption spectra and the M state decay of D96N mutant BR The absorption spectra of D96N mutant BR was examined over the range of 370 to 700 nm in a 1 ml cuvette with a 1.0 em path length. The kinetics of M state decay were measured at 410 nm using a HP 8452A UV/VIS spectrophotometer with a minimum integration time of 100 ms. The light from a Kodak Carousel 35 mm projector, containing a Oriel yellow filter no. 59494 which eliminated wavelength below 520 nm, was focused by a collimating lens and reflected by a mirror down into the cuvette. The light was blocked for dark measurements by placing a piece of black paper between the light source and the cuvette. The kinetic measurement was started in the presence of light passing down through the cuvette, followed by the measurement in the dark. The time re~ quired for the absorbance to reach half way between the steady-state values in the dark and in the light was reported as the half-time (Tij2) and represented the kinetic data for the M state decay. This half-time was calculated by fitting an single exponential function to the absorbance versus time to study the relative variation in the kinetics of the M state decay.
2.3. Preparation of immobilized D96N BR and enzyme in sol-gel glass The sol-gel glass was prepared as previously described [18]. The enzyme was immobilized as follow. The solution of TMOS (TMOS, 7 ml; distilled water, 1 mi; 0.04 M HCI, 0.1 ml) was sonicated for 20 rain. The resulting solutiotl was diluted with an equal volume of distilled water. The diluted TMOS solution (0.5 ml) was added into the 1 ml cuvette followed by the addition of a 2.1 mg mi-t D96N BR, and of enzyme solution of the desired units in 0.1 M borate buffer (pH 9.0). The volumetric ratio of TMOS, BR and enzyme in the cuvette was as follows; TMOS, 0.5 ml; enzyme solution, 0.25 ml and D96N BR, 0.25 mi. The contents of the cuvette were thoroughly mi~ed and allowed to lbrm a gel. The gel formed in this manner was then suspended in the desired buffer for 1 h.
2.4. Measurement of the kinetics of M state decay of D96N BR with immobilized enzymes and BR tn ~o~ogel glass The BR and enzyme immobilized in sol-gel glass were incubated in the working buffer for 2 h prior to the measurement. The gel was then placed into the substrate solution of varying concentrations for 30 rain. The gel was then removed and placed into a I ml cuvette for the
472
P.C. Pandey et al. I Sensors and Actuators B35-36 (1996) 470-474
1.0000 f m 0.60000 o z
< 0.60000
m
/
D:
\X
o 0.40000 ;'"-t._
m
0.20000
_
0.0000
,
~
//
\
'~ , .," ""'" ..,_,,.,~"
-
"%.
.:..~...__.,
\
"%
S
---~~
• I
. . . .
i
toO0
,
,
,
500
I
600
,
~
,
,
I
700
WAVELENGTH
Fig. I. The absorplion spectrum of D96N mutant BR (I) ia the absence of an external light source; the other curves al~ ia th~ p~csence of an external light source; (2) in 0.01 M phosphate h~:f'f,~rfpH 7.0); (3) in ammonium chloride (0.02 M in 0.0l M phosphat~ buffer, pH 7.0) and (4) in benzylamine (0.04 M in 0.01 M phosphate buffer, pH 7.0); (BR.
0.28 mg ml-I). measurement of the kinetics of M state decay. Phosphate buffer (0.01 M, pH 7.0) was used as the working buffer for the measurement of the enzymatically catalyzed reactions. 3. Results and discussion
Several known variants of BR with modified optical properties have been reported. These are based on the exchange of the retinylidene residue for a retinal analog molecule [27] or on the modification of the amino acid sequence of native BR [28]. Tho D96N mutant was used throughout the present investigation. This D96N mutant differs from the wild type by substitution of Asp96 for an Asn residue. This results in a reduction in the internal proton donor capability and a dependence of the M state decay rate on the extracellular pH. Fig, I shows the absorption spectrum of D96N BR in 0.01 M phosphate buffer (pH 7.0), over the range of 370 to 700 nm in the absence of any external light source (curve I)and in the presence of yellow light (curve 2). In the absence of light, a larger peak is observed at 570 nm. The reverse is observed in the presence of light. Curves 3 and 4 show the absorption spectrum in the presence of ammonium chloride and benzylamine, respectively, in the presence of the external light source. There is a decrease in absorption maxima at 570 whereas it increases at 410 that shows the presence of BR in M state. In the presence of ammonium ion (curve 3), less absorbance was observed, whereas the presence of benzylamine (curve 4) resulted in an increase in absorbance, as compared to the absorbance in 0.01 M phosphate buffer (pH 7.0, curve 2). Additionally in the presence of ammonium ion both 410 and 570 nm components arc seen (curve 3). It shows that the lifetime of the M state increases in the presence of benzylamine proba-
bly associated with proton accepter capability of the amino group whereas it decreases in the presence of ammonium ion which is proton rich under similar condition. The overall rate of the M state decay back to trans-BR includes the rate of two subsequent steps: (i) conversien rate of the M state to N state and (it) rate of retinal reisomerization back to trans-BR. The data reported in this paper show the overall rate for the conversion of M state back to trans-BR. Step (i) determines the lifetime of the M state whereas step (it) is dependent on the pH of the medium. Fig. 2 shows the kinetics of M state decay of D96N mutant BR at 410 rim. The formation of the M state takes place in the presence of light, as observed at 410 nm. After 5 s of yellow light exposure, the cuvette was covered with a piece of black paper and the observed decay rate back to groundstate was taken as the M state decay rate. The curves l, 2 and 3 show the kinetics of M state decay in 0.01 M phosphate buffer (pH 7.0), 0.003 M ammonium chloride and 0.04 M benzylamine, respectively, leading to the apparent change in the absorption towards the lowest concentration side. The absorbance maxima increases in the presence of benzylamine (curve 3) whereas it decreases in the presence of ammonium chloride (curve 2) as compared to the absorbance maxima obtained in buffer only (curve 1). The half-time (Tin), which is the time required for the absorhance maxima to decay half way to the absorbance for ground state, were calculated under each set of conditions. Fig. 3 shows the variation of Tir~ as a function of the concentrations of benzyhtmine and ammonium chloride. The Tt12 value changes in opposite directions in these two cases. The reason for this variation is believed to be associated with the different rate for the step (i) of the overall rate which changes in the lifetime of the M state during the photocycle. 1.0000 I ra 080000 C,J
I-.- I
< 0.60000
o(/I OAO000
2
< 0,20000 0,0000
,,,
0
I ....
5
I ....
10
I ....
15
I ....
20
I I ~ i ,I
25
30
SECONDS Fig. 2. The kinetics of M state decay of the D96N mutant at 410 nm; the initial data recorded between 0 and -5 s represent the absorption in light, whereas the rest of the decay curve shows the absorption change in dark, back to the ground state; (1) 0.Ol M phosphate buffer (pH 7.0), (2) 0.003 M ammonium chloride in 0.0l M phosphate buffer (pH 7.0); (3) 0.04 M benzylamine in 0.01 M phosphate buffer (pH 7.0); (BR, 0.31 mg ml-i.
473
P.C. Pandey et al. / Sensors and Actuators B35-36 (1996) 470-474 1.5000
.-
1,2000
--.~-~
~ Z
< 090000
w
#
IN2
o
!
0,60000
......
< 030000 t 0.0000"''' 0
I .... 5
t .... 10
i .... 15
i .... 20
J .... 25
i 30
SECONDS 0
0.00
I
I
0.02
0,0t,
Ammonium
~
0.06
I
I
0.08
0.10
chloride/Benzytomine
(~1
F/g, 3. Variation of the half time (TII 2) of M state decay of D96N mutant BR in the presence of different concentrations of ( I ) ammonium chloride and (2) benzylamine m 0.Ol M phosphate buffer (pH 7.0); (BR, 0,31 mg ml-I).
The time constant of the M state decay, as a function of pH (step (it) of the overall rate) has been explained by several workers [7,8,13,14]. It has been shown [7] that above pH 8.0, the slower M decay component becomes progressively slower with increasing pH. The chromophore reactions remain constant at low and high pH. However, at low pH, the extracellular proton release complex remains protonated throughout, while at high pH, it deprotonates early in the cycle. At low pH, there is an increased rate of retinal reisomerization resulting in rapid decay of the M state to trans-BR. At higher pH, the rate of retinal reisomerization becomes slower, leading to slow decay of the M state. The sharp dependence of the kinetics of M state decay on the proton uptake directed our interest to examine the M state decay in the presence of an enzymatic system that involves the participation of protons. We have observed very interesting results while measuring the M state decay in the presence of either urease, acetylcholinesterase or penieillinase catalyzed enzymatic reactions. Fig. 4 shows the kinetics of M state decay in the presence and absence of an enzymatic system involving urease and acetylcholinesterase. Faster kinetics are observed in the presence of both enzymatic systems while the measurements were made in 0.01 M phosphate buffer (pH 7.0). The pH of the enzymatic reaction mixture was also measured and it was found that there was a decrease in pH from 7 to 5.9 with acetylcholinesterase and to 6.3 with penicillinase whereas the pH was slightly up (7.4) with urease. The faster M state kinetics should be observed with acetylcholinesterase and penicillinase whereas the slower kinetics should be observed with urease based on the dependence of the retinal reisomerization on pH as reported by earlier workers [7,8,13,14,25]. However the data plotted in Fig. 5 show
Fig. 4. The effect~ of the enzymatic reaction systems on the kinetics of M state decay of D96N mutant BR at 410 nm; the initial data recorded between 0 and 5 s represent the absorption in light, whereas the rest of the decay curve shows the absorption change in dark, back to the ground state; (I) 0.01 M phosphate buffer (pH 7.0); (2) in the presence of urease (0.3 mg m1-1) and urea 0.02 M in 0.01 M phosphalt~ buffer (pH 7.0); (3) in presence of acetylcholinesterasc (0.08 mg ml -I) and 0.02 M acetylcholine in 0.01 M phosphate buffer (pH 7.0); (BR, 0.31 mg ml-I).
that in all cases the M state decay kinetics becomes faster as a result of the enzymatic reaction. This observation shows that the ammonium ion formed as a result of the urease catalyzed reaction is causing a decrease in the lifetime of the M sta~e that results in a decay rate faster back to trans-BR. It can be concluded from these observations that step (i) (rate of the conversion of the M state to N state) is the primary determining step of the overall rate of decay of the M state back to trans-BR. The kinetics are
' ¢: o O t/) ! IN
Ib,- . . . .
0 .... 0.00
1-
I 0.02
-o-
t 0.0/,
Urea/Acetylchollne
_ t 0.06
E!....... 0.08 0.10
/ Penicillin
{M)
Fig. 5. Variation of the half time (TIJ2) of M state decay of D96N mutant BR in the presence of three enzymatic systems: (I) urea, (2) acetylcholine and (3) penicillin.
474
P.C Pandey et al. / Sensors and Actuators B35-36 (1996) 470-474
unaffected in the absence of substrate or in the presence oi"enzyme alone. The present research describes the variation in the kinetics of M state decay to ground state. The formation of the M state takes place when bacteriorhodopsin is exposed to yellow light. The rate of decay in the dark increases in the presence of ammonium ion associated with the decrease in the lifetime of the M state whereas, in the presence of an aromatic amine, it decreases due to increase in the lifetime of the same. This dependence of the M state decay rate on the lifetime of the M state and subsequent dependence of retinal reisomerization on the pH of the medium has been exploited in probing the enzymatic catalyzed reactions using urease, acetylcholinesterase and peniciilinase. References [1] J.K. Delaney, T.L. Brack, G.H. Alkinson, M. Ottolenghi. N. Friedman and M. Sheves, J. Phys, Chem., 97 (1993) 1241612422. [2] J.Y. Huan8, Z.P. Chen and A. Lewis, J. Phys. Chem.. 93 (1989) 3814. [3] M,P. Krebs, W. Behrens, R. Mollaaghababa, H.G. Khorana and M.P. Heyn, Biochemistry, 32 (1993) 12830-.12834. [4] K.J. Rothschild, T. Mufti, S. Sonar, Y.W. He, P. Rath, W. Fischer and H.G, Khorana, J. Biol, Chert, 268 (1993) 27046-27052. [5] L.A, Droehev, A.D. Kaulen and A.Y. Komrokov, Biochem. Mol. Biol. Int., 30 (1993)461-469.
[6] S.P. Balashov, R. Govindjee, M. Kono, E. Imasheva, E. Lukashev, T.G. Ebrey, R.K. Crouch, D.R. Menick and Y. Feng, Biochemistry. 32 (1993) 10331-10343. [7] Y. Cao, L.S. Brown, R. Needleman and J.K. Lanyi, Biochemistry, 32 (1993) 10239-10248. [8] I~. Zimanyi, Y. Can, R. Needlema~, M. Ottolenghi and J.K. Latlyi, Biochemls;ry, 32 (I 993) 7669-7678,
[9] L.S. Brown, L. Zimanyi, R. Needleman, M. Ottolenghi and J.K. ~.~a'tyi,Biochemistry, 32 (1993) 7679-7685. [10] F. Zhou, A. Windemuth and K. Schulten, Biochemistry, 32 (1993) 2291-2306. [11] Y. Cao, G. Varo, A.L. Klinger, D.M. Czajkowsky, M.S. Braiman, R. Needleman and J.K. Lanyi, Biochemistry. 32 (1993) 19811990. [12] S.G. Wu, L.M. EUerby, J.S. Cohan, B. Dunn and M.A. Elsayed, Chem. Mater., 5 (1993) 115-120. [13] L.A. Drochev, S.V. Dracheva and A.D. Kanlen, FEBS Lett., 332 (1993) 67-70. [14] M. Kono, S. Misro and T.G. Ebrey, FEBS Lett., 331 (1993) 3134. [15] K.C. Chou, J. Protein Chem., 3 (1993) 337-350. [16] Y. Sben, C.R. Safinya, K.S. Liang, A.F. Ruppert and K.J. Rothschild, Nature, 366 (1993) 48-50. [17] Y.N. Zhang, M,A, EIsayed, L.J. Stern, T. Marti, T. Mogi and H.G. Khorona Photochem. Photobiol.. 57 (1993) 1027-103 I. [18] H.H. Weetall, B. Robenson, D. Cullin, J. Brown anO M. Walch, Biochim. Biophys. Acta, 1142 (1993) 211-213. [19] J,K. Lanyi, Experientia, 49(1993) 514-517. [20] L.S. Brown, Y, Yamazaki, A. Maeda, L. Su, R. Needleman and J.K. Lanyi, Biophys. J., 66 (1993) AI. [21] R. Govindjee, S.P. Balashov, T.G. Ebrey, D. Oesterbelt, G. Steinberg and Sbeves, M., Biophys. J., 66 0993) A45. [22] R.V. Sampogna, A.S. Yang, and B.H. Honig,. Biophys. J., 66 (1993) A45. [23] D. Oesterhelt and W. Stoeckenius, Nature. 233 (1971) 149-152. [24] D. Oesterhelt, J. Tittor and E.J. Bamberg, Bioeenerg. Biomembr., 24 (1992) 181-191. [25] M. Nakasako, M. Katanka and F. Tokunaga, FEBS Lett., 254 (1989) 215-218. [26] M. Yoshida, K. Ohono, Y. Takeuchi and Y. Kagawa, Biochem. Biophys. Res. Commun., 75 (1977) I I I l - i I IS. [27] J. Zingoni, R K. Crouch, C.H. Chijang. R Govindjee and T.G. Ebrey, Biochemistry, 25 0986) 2022. [28] J. Soppa and D. Oesterbelt, J. Biol. Chen~. 264 (1989) 13043.
ELSEVIER
Sensorsand ActuatorsB 35-36 (1996) 470--474
eHe-fi'~V~Zt
Reversal in the kinetics of the M state decay of D96N bacteriorhodopsin: probing of enzyme catalyzed reactions P.C. P a n d e y a,*, S u d h a S i n g h a, B i p i n U p a d h y a y a, H o w a r d H. W e e t a U b, P e t e r K. C h e n c aDepartmen¢of Chemistry, BanarasHindu University, Varanasi221005, India bBiotechnology Division. NationalInstituteof Standards and Technology. Gaithersburg. MD 20899. USA cDelmrfmentof Biology, GeorgetownUniversity, Washington,DC 20057, USA
Abstract The kinetics of M state decay of the D96N bacteriorhodopsin (BR) in the presence of (i) ammonium ion and (ii) an aromatic amine is reported. The M state decay rate becomes faster in the presence of ammonium salt with a concomitant decrease in absorbance maxima at 410 nm. in the presence of an aromatic amine, a slower M state decay rate with an increase in absorbance maxima at 410 nm is observed. The variation in the kinetics of M-state decay is due to the change in the lifetime of the M-state associated with the conversion of M-intermediate to trans-BR. The decay rate of the M-state has also been found to be greatly affected in the presence of enzymatic reactions resulting in the formation of either an acid or a base. Three enzymatic reaction systems, urease, acetylcholinesterase and penicillinase, have been used to study the kinetics of M state decay of D96N bacteriorhodopsin. The kinetic data are found to be dependent on the concentrations of the respective enzyme's substrate. Results of the studies using urea, acetylcholine and penicillin are reported. The enzymes were also immobilized together with D96N bacteriorhodopsin in sol-gel glass to develop a biosensor for the analysis of urea, acetylcholine and penicillin. Keywords: M state decay; Kinetics; D96N bacteriorhodopsin
1, Intredection Over the last few years [1-22] there has been increased attention focused on the study of the photoactivity of bacteriorhodopsin (BR). The purple membrane protein of Haiobacterium solinarium (previously Halobacterium halobium) acts as a proton-pump after photoexcitation, transporting protons from the cytoplasmic side of the membrane to the extracellular environment against a concentration gradient [10,23]. The photocycle consists of a series of steps which are initiated on absorption of a photon. The kinetics of photoexcitation ultimately determines the behavior of proton-pumping and light absorption efficiency of the bacteriorhodopsin (BR). The proton pumping results in a pH gradient based on directed sequential changes of the pKa of the retinal Schiff base [11.24]. During the photocycle, the proton translocation is accomplished by the deprotonation of a Schiff base followed by * Conespondingauthor.
ElsevierSdenceS.A. PII
S0925-4005(96)02083-7
reprotonation. The deprotonation step produces the M state intermediate from the K and L intermediates which are formed subsequently once retinal is isomerized in the presence of an external light source. The reprotonation initiates the M state decay in the absence of an external light source. There are several reports [7,8,11] describing proton uptake in bacteriorhodopsin. Retinal isomerization reactions are important in the proton pumping cycle of bacteriorhodopsin [10]. The retinal undergoes an all-trans 13-cis or an all-trans-~ 13,14-di-cis photoisomerization as a very fast primary step in the photocycle forming the K intermediate. The initial retinal isomerization step, although very fast (3 ps), has been found to be dependent on internal water molecules [10]. The water affects the nature of the Schiff base counter ion and the nature of the primary photoreaction. It was suggested that water chains subsequently effect the reprotonation and deprotonation steps of the proton pump photocycle [10]. The deprotonation of L state (an intermediate formed from the K in-
P.C. Pandey et al. / Sensors and Actuators 1735-36 (1996) 470--474
termediate) leads to the formation of M state which decays back to trans-BR in the absence of an external light source. The presence of the M state can be monitored by the measurement of the absorption at 410 nm. It has been observed that the lifetime of the Mintermediate in the BR photocycle was remarkably prolonged on drying a suspension of purple membrane [25] in the presence of arginine. Guanidine has also been reported to increase the lifetime of M state of BR in a similar fashion [26]. It is important to understand the effects of the proton donor/acceptor present on the extracellular side with respect to the lifetime of the M state. The lifetime of the M state eventually determines decay rate back to the ground state (trans-BR) after reisomerization in the dark which is a slower step of the photocycle. We chose to use ammonium ion and a primary aromatic amine to modify the kinetics of M state decay since these molecules may effect the lifetime of the M state in the photocycle. The effect of pH on the retinal reisomerization has been reported by several workers [7,8,13,141, At low pH, the retinal reisomerization occurs first, whereas at high pH, this is delayed. The delay in retinal reisomerization results in a slower decay of the M state. The pH dependence of retinal reisomerization directed our attention to study the decay rate of the M state in the presence of the enzymatic systems leading to the formation of either an acid or a base. This type of reaction will affect the kinetics of M state decay of the D96N mutant BR. This change in the kinetics of the M state decay can thus be used in monitoring the enzymatic reactions associated with the formation of either an acid or a base. This relationship may he useful in the development of future BR containing analytical devices. In this payer we report the kinetics of the M state decay of DetiN BR in the presence of ammonium salt and benzylamine. The enzymatic reactions employing urease, acetylcholinesterase and peniciUinase has also been monitored, based on the measurement of the kinetics of the M state decay in the dark. These enzymes together with bacteriorhodopsin have been immobilized in a solgel glass as a first step in the development of a biosensor for the analysis of urea, acetyicholine and penicillin.
2. Experimental 2.1. Materials The materials used in this investigation and their sources were as follows: The D96N mutant bacteriorhodopsin used in this investigation was supplied by Dr A.B. Druzhko0 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchina, Russia. Urease, acetylcholinesterase, penicillinase, benzylamine, penicillin G, acetylcho|ine were obtained form Sigma Chemical Company (St. Louis, MO); Tetrameth-
471
oxy-silane (TMOS) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). All other chemicals used were of analytical grade.
2.2. Measurement of absorption spectra and the M state decay of D96N mutant BR The absorption spectra of D96N mutant BR was examined over the range of 370 to 700 nm in a 1 ml cuvette with a 1.0 em path length. The kinetics of M state decay were measured at 410 nm using a HP 8452A UV/VIS spectrophotometer with a minimum integration time of 100 ms. The light from a Kodak Carousel 35 mm projector, containing a Oriel yellow filter no. 59494 which eliminated wavelength below 520 nm, was focused by a collimating lens and reflected by a mirror down into the cuvette. The light was blocked for dark measurements by placing a piece of black paper between the light source and the cuvette. The kinetic measurement was started in the presence of light passing down through the cuvette, followed by the measurement in the dark. The time re~ quired for the absorbance to reach half way between the steady-state values in the dark and in the light was reported as the half-time (Tij2) and represented the kinetic data for the M state decay. This half-time was calculated by fitting an single exponential function to the absorbance versus time to study the relative variation in the kinetics of the M state decay.
2.3. Preparation of immobilized D96N BR and enzyme in sol-gel glass The sol-gel glass was prepared as previously described [18]. The enzyme was immobilized as follow. The solution of TMOS (TMOS, 7 ml; distilled water, 1 mi; 0.04 M HCI, 0.1 ml) was sonicated for 20 rain. The resulting solutiotl was diluted with an equal volume of distilled water. The diluted TMOS solution (0.5 ml) was added into the 1 ml cuvette followed by the addition of a 2.1 mg mi-t D96N BR, and of enzyme solution of the desired units in 0.1 M borate buffer (pH 9.0). The volumetric ratio of TMOS, BR and enzyme in the cuvette was as follows; TMOS, 0.5 ml; enzyme solution, 0.25 ml and D96N BR, 0.25 mi. The contents of the cuvette were thoroughly mi~ed and allowed to lbrm a gel. The gel formed in this manner was then suspended in the desired buffer for 1 h.
2.4. Measurement of the kinetics of M state decay of D96N BR with immobilized enzymes and BR tn ~o~ogel glass The BR and enzyme immobilized in sol-gel glass were incubated in the working buffer for 2 h prior to the measurement. The gel was then placed into the substrate solution of varying concentrations for 30 rain. The gel was then removed and placed into a I ml cuvette for the
472
P.C. Pandey et al. I Sensors and Actuators B35-36 (1996) 470-474
1.0000 f m 0.60000 o z
< 0.60000
m
/
D:
\X
o 0.40000 ;'"-t._
m
0.20000
_
0.0000
,
~
//
\
'~ , .," ""'" ..,_,,.,~"
-
"%.
.:..~...__.,
\
"%
S
---~~
• I
. . . .
i
toO0
,
,
,
500
I
600
,
~
,
,
I
700
WAVELENGTH
Fig. I. The absorplion spectrum of D96N mutant BR (I) ia the absence of an external light source; the other curves al~ ia th~ p~csence of an external light source; (2) in 0.01 M phosphate h~:f'f,~rfpH 7.0); (3) in ammonium chloride (0.02 M in 0.0l M phosphat~ buffer, pH 7.0) and (4) in benzylamine (0.04 M in 0.01 M phosphate buffer, pH 7.0); (BR.
0.28 mg ml-I). measurement of the kinetics of M state decay. Phosphate buffer (0.01 M, pH 7.0) was used as the working buffer for the measurement of the enzymatically catalyzed reactions. 3. Results and discussion
Several known variants of BR with modified optical properties have been reported. These are based on the exchange of the retinylidene residue for a retinal analog molecule [27] or on the modification of the amino acid sequence of native BR [28]. Tho D96N mutant was used throughout the present investigation. This D96N mutant differs from the wild type by substitution of Asp96 for an Asn residue. This results in a reduction in the internal proton donor capability and a dependence of the M state decay rate on the extracellular pH. Fig, I shows the absorption spectrum of D96N BR in 0.01 M phosphate buffer (pH 7.0), over the range of 370 to 700 nm in the absence of any external light source (curve I)and in the presence of yellow light (curve 2). In the absence of light, a larger peak is observed at 570 nm. The reverse is observed in the presence of light. Curves 3 and 4 show the absorption spectrum in the presence of ammonium chloride and benzylamine, respectively, in the presence of the external light source. There is a decrease in absorption maxima at 570 whereas it increases at 410 that shows the presence of BR in M state. In the presence of ammonium ion (curve 3), less absorbance was observed, whereas the presence of benzylamine (curve 4) resulted in an increase in absorbance, as compared to the absorbance in 0.01 M phosphate buffer (pH 7.0, curve 2). Additionally in the presence of ammonium ion both 410 and 570 nm components arc seen (curve 3). It shows that the lifetime of the M state increases in the presence of benzylamine proba-
bly associated with proton accepter capability of the amino group whereas it decreases in the presence of ammonium ion which is proton rich under similar condition. The overall rate of the M state decay back to trans-BR includes the rate of two subsequent steps: (i) conversien rate of the M state to N state and (it) rate of retinal reisomerization back to trans-BR. The data reported in this paper show the overall rate for the conversion of M state back to trans-BR. Step (i) determines the lifetime of the M state whereas step (it) is dependent on the pH of the medium. Fig. 2 shows the kinetics of M state decay of D96N mutant BR at 410 rim. The formation of the M state takes place in the presence of light, as observed at 410 nm. After 5 s of yellow light exposure, the cuvette was covered with a piece of black paper and the observed decay rate back to groundstate was taken as the M state decay rate. The curves l, 2 and 3 show the kinetics of M state decay in 0.01 M phosphate buffer (pH 7.0), 0.003 M ammonium chloride and 0.04 M benzylamine, respectively, leading to the apparent change in the absorption towards the lowest concentration side. The absorbance maxima increases in the presence of benzylamine (curve 3) whereas it decreases in the presence of ammonium chloride (curve 2) as compared to the absorbance maxima obtained in buffer only (curve 1). The half-time (Tin), which is the time required for the absorhance maxima to decay half way to the absorbance for ground state, were calculated under each set of conditions. Fig. 3 shows the variation of Tir~ as a function of the concentrations of benzyhtmine and ammonium chloride. The Tt12 value changes in opposite directions in these two cases. The reason for this variation is believed to be associated with the different rate for the step (i) of the overall rate which changes in the lifetime of the M state during the photocycle. 1.0000 I ra 080000 C,J
I-.- I
< 0.60000
o(/I OAO000
2
< 0,20000 0,0000
,,,
0
I ....
5
I ....
10
I ....
15
I ....
20
I I ~ i ,I
25
30
SECONDS Fig. 2. The kinetics of M state decay of the D96N mutant at 410 nm; the initial data recorded between 0 and -5 s represent the absorption in light, whereas the rest of the decay curve shows the absorption change in dark, back to the ground state; (1) 0.Ol M phosphate buffer (pH 7.0), (2) 0.003 M ammonium chloride in 0.0l M phosphate buffer (pH 7.0); (3) 0.04 M benzylamine in 0.01 M phosphate buffer (pH 7.0); (BR, 0.31 mg ml-i.
473
P.C. Pandey et al. / Sensors and Actuators B35-36 (1996) 470-474 1.5000
.-
1,2000
--.~-~
~ Z
< 090000
w
#
IN2
o
!
0,60000
......
< 030000 t 0.0000"''' 0
I .... 5
t .... 10
i .... 15
i .... 20
J .... 25
i 30
SECONDS 0
0.00
I
I
0.02
0,0t,
Ammonium
~
0.06
I
I
0.08
0.10
chloride/Benzytomine
(~1
F/g, 3. Variation of the half time (TII 2) of M state decay of D96N mutant BR in the presence of different concentrations of ( I ) ammonium chloride and (2) benzylamine m 0.Ol M phosphate buffer (pH 7.0); (BR, 0,31 mg ml-I).
The time constant of the M state decay, as a function of pH (step (it) of the overall rate) has been explained by several workers [7,8,13,14]. It has been shown [7] that above pH 8.0, the slower M decay component becomes progressively slower with increasing pH. The chromophore reactions remain constant at low and high pH. However, at low pH, the extracellular proton release complex remains protonated throughout, while at high pH, it deprotonates early in the cycle. At low pH, there is an increased rate of retinal reisomerization resulting in rapid decay of the M state to trans-BR. At higher pH, the rate of retinal reisomerization becomes slower, leading to slow decay of the M state. The sharp dependence of the kinetics of M state decay on the proton uptake directed our interest to examine the M state decay in the presence of an enzymatic system that involves the participation of protons. We have observed very interesting results while measuring the M state decay in the presence of either urease, acetylcholinesterase or penieillinase catalyzed enzymatic reactions. Fig. 4 shows the kinetics of M state decay in the presence and absence of an enzymatic system involving urease and acetylcholinesterase. Faster kinetics are observed in the presence of both enzymatic systems while the measurements were made in 0.01 M phosphate buffer (pH 7.0). The pH of the enzymatic reaction mixture was also measured and it was found that there was a decrease in pH from 7 to 5.9 with acetylcholinesterase and to 6.3 with penicillinase whereas the pH was slightly up (7.4) with urease. The faster M state kinetics should be observed with acetylcholinesterase and penicillinase whereas the slower kinetics should be observed with urease based on the dependence of the retinal reisomerization on pH as reported by earlier workers [7,8,13,14,25]. However the data plotted in Fig. 5 show
Fig. 4. The effect~ of the enzymatic reaction systems on the kinetics of M state decay of D96N mutant BR at 410 nm; the initial data recorded between 0 and 5 s represent the absorption in light, whereas the rest of the decay curve shows the absorption change in dark, back to the ground state; (I) 0.01 M phosphate buffer (pH 7.0); (2) in the presence of urease (0.3 mg m1-1) and urea 0.02 M in 0.01 M phosphalt~ buffer (pH 7.0); (3) in presence of acetylcholinesterasc (0.08 mg ml -I) and 0.02 M acetylcholine in 0.01 M phosphate buffer (pH 7.0); (BR, 0.31 mg ml-I).
that in all cases the M state decay kinetics becomes faster as a result of the enzymatic reaction. This observation shows that the ammonium ion formed as a result of the urease catalyzed reaction is causing a decrease in the lifetime of the M sta~e that results in a decay rate faster back to trans-BR. It can be concluded from these observations that step (i) (rate of the conversion of the M state to N state) is the primary determining step of the overall rate of decay of the M state back to trans-BR. The kinetics are
' ¢: o O t/) ! IN
Ib,- . . . .
0 .... 0.00
1-
I 0.02
-o-
t 0.0/,
Urea/Acetylchollne
_ t 0.06
E!....... 0.08 0.10
/ Penicillin
{M)
Fig. 5. Variation of the half time (TIJ2) of M state decay of D96N mutant BR in the presence of three enzymatic systems: (I) urea, (2) acetylcholine and (3) penicillin.
474
P.C Pandey et al. / Sensors and Actuators B35-36 (1996) 470-474
unaffected in the absence of substrate or in the presence oi"enzyme alone. The present research describes the variation in the kinetics of M state decay to ground state. The formation of the M state takes place when bacteriorhodopsin is exposed to yellow light. The rate of decay in the dark increases in the presence of ammonium ion associated with the decrease in the lifetime of the M state whereas, in the presence of an aromatic amine, it decreases due to increase in the lifetime of the same. This dependence of the M state decay rate on the lifetime of the M state and subsequent dependence of retinal reisomerization on the pH of the medium has been exploited in probing the enzymatic catalyzed reactions using urease, acetylcholinesterase and peniciilinase. References [1] J.K. Delaney, T.L. Brack, G.H. Alkinson, M. Ottolenghi. N. Friedman and M. Sheves, J. Phys, Chem., 97 (1993) 1241612422. [2] J.Y. Huan8, Z.P. Chen and A. Lewis, J. Phys. Chem.. 93 (1989) 3814. [3] M,P. Krebs, W. Behrens, R. Mollaaghababa, H.G. Khorana and M.P. Heyn, Biochemistry, 32 (1993) 12830-.12834. [4] K.J. Rothschild, T. Mufti, S. Sonar, Y.W. He, P. Rath, W. Fischer and H.G, Khorana, J. Biol, Chert, 268 (1993) 27046-27052. [5] L.A, Droehev, A.D. Kaulen and A.Y. Komrokov, Biochem. Mol. Biol. Int., 30 (1993)461-469.
[6] S.P. Balashov, R. Govindjee, M. Kono, E. Imasheva, E. Lukashev, T.G. Ebrey, R.K. Crouch, D.R. Menick and Y. Feng, Biochemistry. 32 (1993) 10331-10343. [7] Y. Cao, L.S. Brown, R. Needleman and J.K. Lanyi, Biochemistry, 32 (1993) 10239-10248. [8] I~. Zimanyi, Y. Can, R. Needlema~, M. Ottolenghi and J.K. Latlyi, Biochemls;ry, 32 (I 993) 7669-7678,
[9] L.S. Brown, L. Zimanyi, R. Needleman, M. Ottolenghi and J.K. ~.~a'tyi,Biochemistry, 32 (1993) 7679-7685. [10] F. Zhou, A. Windemuth and K. Schulten, Biochemistry, 32 (1993) 2291-2306. [11] Y. Cao, G. Varo, A.L. Klinger, D.M. Czajkowsky, M.S. Braiman, R. Needleman and J.K. Lanyi, Biochemistry. 32 (1993) 19811990. [12] S.G. Wu, L.M. EUerby, J.S. Cohan, B. Dunn and M.A. Elsayed, Chem. Mater., 5 (1993) 115-120. [13] L.A. Drochev, S.V. Dracheva and A.D. Kanlen, FEBS Lett., 332 (1993) 67-70. [14] M. Kono, S. Misro and T.G. Ebrey, FEBS Lett., 331 (1993) 3134. [15] K.C. Chou, J. Protein Chem., 3 (1993) 337-350. [16] Y. Sben, C.R. Safinya, K.S. Liang, A.F. Ruppert and K.J. Rothschild, Nature, 366 (1993) 48-50. [17] Y.N. Zhang, M,A, EIsayed, L.J. Stern, T. Marti, T. Mogi and H.G. Khorona Photochem. Photobiol.. 57 (1993) 1027-103 I. [18] H.H. Weetall, B. Robenson, D. Cullin, J. Brown anO M. Walch, Biochim. Biophys. Acta, 1142 (1993) 211-213. [19] J,K. Lanyi, Experientia, 49(1993) 514-517. [20] L.S. Brown, Y, Yamazaki, A. Maeda, L. Su, R. Needleman and J.K. Lanyi, Biophys. J., 66 (1993) AI. [21] R. Govindjee, S.P. Balashov, T.G. Ebrey, D. Oesterbelt, G. Steinberg and Sbeves, M., Biophys. J., 66 0993) A45. [22] R.V. Sampogna, A.S. Yang, and B.H. Honig,. Biophys. J., 66 (1993) A45. [23] D. Oesterhelt and W. Stoeckenius, Nature. 233 (1971) 149-152. [24] D. Oesterhelt, J. Tittor and E.J. Bamberg, Bioeenerg. Biomembr., 24 (1992) 181-191. [25] M. Nakasako, M. Katanka and F. Tokunaga, FEBS Lett., 254 (1989) 215-218. [26] M. Yoshida, K. Ohono, Y. Takeuchi and Y. Kagawa, Biochem. Biophys. Res. Commun., 75 (1977) I I I l - i I IS. [27] J. Zingoni, R K. Crouch, C.H. Chijang. R Govindjee and T.G. Ebrey, Biochemistry, 25 0986) 2022. [28] J. Soppa and D. Oesterbelt, J. Biol. Chen~. 264 (1989) 13043.