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Radiation inactivation study of aminopeptidase: probing the active site
ARTICLE IN PRESS
Radiation Physics and Chemistry 69 (2004) 473–479
Radiation inactivation study of aminopeptidase: probing the active site V.K. Jamadara, S.N. Jamdara, Hari Mohanb, S.P. Dandekarc, P. Harikumara,* a
Food Technology Division, Flesh Food Biochemistry Section, BARC, Mumbai 400 085, India b Radiation Chemistry & Chemical Dynamics Division, BARC, Mumbai 400 085, India c Department of Biochemistry, Seth G. S. Medical College, KEM Hospital, Mumbai 400 012, India Received 8 April 2003; accepted 30 September 2003
Abstract Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N2O>N2>air. The D37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N2O. The radicals (SCN) 2 , Br 2 and I2 inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiationinduced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies. r 2003 Elsevier Ltd. All rights reserved. Keywords: Radiation inactivation; Aminopeptidase; Pulse radiolysis; Free radicals
1. Introduction Radiation-induced free radicals interact strongly with biomolecules such as DNA (Saha, 2001), proteins (Levin and Roselli, 2001), lipids and other membrane components (Stark, 1991). Enzymes being the core functional units, their inactivation bears a significant impact on the cell metabolism (Saha et al., 1995). Hence, the radiation chemistry of proteins and enzymes assumes paramount importance. From the radiation biology point of view, the free radical chemistry of enzymes has a longstanding interest since such studies give information about the properties of the active site and migration of free radicals through macromolecules (von Sonntag, 1987). The radiation-induced effects have been carried out with major emphasis on the enzymes involved in oxidation– *Corresponding author. Tel.: +91-99224404949; fax: +91225505151. E-mail address: [email protected] (P. Harikumar).
reduction reactions (Saha et al., 1991; Buchanan and Armstrong, 1976, 1978; Abu El Faliat et al., 1983). Among proteases, papain (Clement et al., 1974), trypsin (Masuda et al., 1971; Cudina and Jovanovic, 1988) and carboxypeptidase (Roberts, 1973) have been well studied for their response to ionizing radiation. However, very few efforts have been directed towards understanding the radiation chemistry of N terminal cleaving exopeptidases viz. aminopeptidases. Aminopeptidases (E.C. 3.4.11. ) are a major class of exopeptidases implicated in several pathological conditions (Hui et al., 1995; Preito et al., 1998; Fujimura et al., 2000; Tomoharu et al., 2001) and in general cellular homeostasis (Taylor, 1993). This class of enzymes also finds extensive application in the food industries related to cheese ripening, dairy products and preparation and processing of protein hydrolysates (Flores et al., 1997; Bouchier et al., 2001). Information on the structure–function relationship of aminopeptidases has not been easily forthcoming due to their lability and multimeric
0969-806X/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2003.09.006
ARTICLE IN PRESS V.K. Jamadar et al. / Radiation Physics and Chemistry 69 (2004) 473–479
structural features (Taylor, 1993). In the present study, an attempt has been made to evaluate the influence of gamma radiation and pulse radiolysis as a tool to probe into the active site composition of an aminopeptidase purified to homogeneity (Jamadar et al., 2003) from chicken intestine. Chicken intestine is an important byproduct of the poultry industry, currently being exploited as a rich source of proteins and proteolytic enzymes in our laboratory.
2. Experimental l-arginine-b-naphthylamide (ANA) and mersalyl acid were obtained from Sigma Co., USA. All other chemicals were of Analar grade. N2 and N2O gases used were of IOLAR grade (Indian Oxygen Ltd.) having a purity of X99.9%. The activity of aminopeptidase was determined by the method of Barrett and Kirchke (1981). The assay was carried out at 50 C in 0.1 mol dm3 phosphate buffer pH 6.0 containing 1 103 mol dm3 cysteine using 2 102 mol dm3 ANA as a substrate. The reaction was terminated by addition of 1 103 mol dm3 mersalyl reagent and the pink colour complex was read at 520 nm. Enzyme kinetics including Km ; Vmax and kcat were determined before and after irradiation using the Lineweaver– Burke’s Plot. Aqueous solutions of aminopeptidase (5 103 mol dm3) in 0.25 mol dm3 NaCl and 1 102 mol dm3 phosphate buffer pH 7.0 were irradiated using a Gamma cell 220. The dose rate was 8.5 Gy min1 as determined by Fricke dosimetry (McLaughlin et al., 1989). A 7 MeV linear accelerator (Forward Industries, UK) with a single pulse of 50 ns was used for pulse radiolysis experiments. The detailed working of the system was as described by Guha et al. (1987). The dose rate was determined according to Fielden (1984). For studying the OH radical reactions, the enzyme solution was flushed with N2O gas (IOLAR grade, purity X 99.9%.), to convert all the e aq to OH radicals (Buxton, 1987). The absorption and fluorescence spectra were recorded using the Hitachi 330 spectrometer and Hitachi F 4010 spectrofluorimeter, respectively. The fluorescence lifetime of the enzyme was determined on an EI 199 fluorescence spectrometer (Edinburg, U.K) using the time-correlated single photon counting technique (Pal et al., 1990). The D37 value of the enzyme was determined as the dose required to bring about 63% inactivation of the enzyme. G value was calculated as the number of enzyme molecules inactivated per 100 eV of energy absorbed, and was reported in micromoles/joule (dose rate=8.5 Gy min1) from the slope of the graph plotting D37 vs enzyme concentration (Sanner and Pihl,
1969). The values of percentage inactivation of enzyme are in the range of78–10%.
3. Results and discussion 3.1. Influence of graded dose and irradiation conditions The influence of absorbed radiation dose and conditions of irradiation on aminopeptidase is presented in Fig. 1 and Table 1, respectively. As reported for other enzymes (Sui, 1973; Roberts, 1973), the radiation inactivation of aminopeptidase exhibited an exponential relationship with radiation dose. The exponential relationship observed for aminopeptidase could be due to the non discriminatory interaction of radiolytic free radicals with the active and inactivated enzyme molecules, as pointed out by Sanner and Pihl (1969), for enzymes in general. Data on the influence of different gases in the irradiation media (Table 1) showed that the inactivation was in the order N2O>N2>air with D37 values of 198, 210 and 281 Gy, respectively, indicating thereby that the enzyme inactivation is predominantly OH mediated. The relatively lower effect in the aerated medium as compared to anaerobic conditions could be
10 0
% Residual activity
474
80
60
40
20
0
100
200
300
400
Dose (Gy)
Fig. 1. Effect of absorbed radiation dose on aminopeptidase. Purified aminopeptidase (17 mg/ml) in 0.25 mol dm3 NaCl in 1 102 mol dm3 phosphate buffer pH 7.0 was subjected to g-radiation. The residual enzyme activity was determined under standard assay conditions.
Table 1 D37 and G values of aminopeptidase
D37 (Gy) G value (mmoles/Joule)
Air
N2
N2O
281 0.011
210 0.018
198 0.024
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d
H
þO2 -HOd2 ;
d e aq þO2 -O2 ;
10
1 1
3
k ¼ 2 10 dm mol 10
3
k ¼ 1:9 10 dm mol
s ;
1 1
s ;
ð1Þ ð2Þ
Absence of OH scavenger Isopropanol t-Butanol
120
100
Residual Activity (%)
ascribed, as suggested by Spinks and Woods (1976), to the scavenging of e aq and H atoms by dissolved oxygen, which results in the formation of superoxide anion (O 2 ) and hydroperoxyl radical (HO2 ), respectively (reactions (1)–(3)). These radicals react much slowly compared toOH.
475
80
60
40
HOd2 ¼ Hþ þOd 2 ;
pK ¼ 4:8:
ð3Þ
The G value of aminopeptidase in media saturated with N2O was double that observed in air because e aq is converted to OH radicals (reaction (4)). d e aq þN2 O- OH þ OH þN2 ;
k ¼ 0:91 1010 dm3 mol1 s1 ;
ð4Þ
This is in agreement with the observations of Buchanan and Armstrong (1976) for lactate dehydrogenase. As reported by Winchester and Lynn (1970) for the dipeptide try–gly (Go0:1), G value between 0.01 and 0.03 observed for aminopeptidase (Table 1) could be due to the conversion of tryptophan residues in the enzyme to 3-OH kynurenin or DOPA. However, aminopeptidase being a multimeric polypeptide, the contribution by other residues cannot be ruled out. 3.2. Effect of OH scavengers The role of OH radicals in the inactivation of aminopeptidase was confirmed by studies employing t-butanol and isopropanol which are reported to be scavengers of OH (Nagrani and Bisby, 1989; Adams et al., 1969). Isopropanol scavenges both H, and OH (reactions (5) and (6)). ðCH3 Þ2 CHOH þd OH-H2 O þ ðCH3 Þ2 Cd OH; k ¼ 2 1010 dm3 mol1 s1 :
ð5Þ
ðCH3 Þ2 CHOH þd H-H2 þðCH3 Þ2 C OH; k ¼ 1 108 dm3 mol1 s1 :
ð6Þ
Isopropanol and t-butanol elicited 80% and 95% protection, respectively, to aminopeptidase against radiation-induced inactivation (Fig. 2). This observation reaffirms our contention that the radiation inactivation of this enzyme is predominantly caused by OH radicals. In the case of t-butanol, although H atoms may be interacting with the enzyme, it apparently does not lead to a change in enzyme activity. 3.3. Effect of single electron oxidants on aminopeptidase Radiolytic radicals such as H, OH and e aq could react at random on several sites of a protein but all
20
0
100
200
300
400
Dose (Gy)
Fig. 2. Radiation sensitivity of aminopeptidase in the presence of OH scavengers. Purified aminopeptidase was subjected to gamma radiation in the presence of 1 103 mol dm3 t-butanol and isopropanol. The enzyme solution was purged with N2O gas before irradiation.
reactions need not lead to inactivation. Hence, changes in the enzyme activity per se by these radicals do not point to the specific sites of interactions (Bisby et al., 1974). Hence it would be interesting to use single electron oxidants (viz. Br and (SCN) 2 , I2 2 ) and biochemical modifiers (DMHNB-[Dimethyl (2-hydroxy5-nitrobenzyl) sulfonium bromide]) to gain a better insight into the radiation-sensitive residues in the enzyme. These inorganic radicals are known to react with specific amino acids (Adams et al., 1972). At neutral pH, Br reacts with tyrosine, tryptophan, 2 histidine and cysteine, (SCN) 2 reacts with tryptophan, tyrosine, cysteine and methionine while I reacts only 2 with cysteine (Hasan et al., 1994). These chemical probes can thus be used to identify the radiation-sensitive residues in the enzyme which may be crucial to its catalytic function. The data presented in (Fig. 3) show that gamma radiation (400 Gy) inactivated aminopepti dase by 96%, 85% and 80% in I 2 , (SCN)2 and Br2 , respectively. The observation that the enzyme was completely inactivated in the presence of the above radicals suggests the involvement of aromatic and thiol amino acids in the catalytic activity of the enzyme. The highest effectiveness of I 2 points to the participation of cysteine residues. In fact our earlier studies using cysteine, 2-mercaptoethanol and dithiothreitol have shown that the number of available SH residues is a critical factor in eliciting the activity of the enzyme (Jamadar et al., 2003). The decisive role of tryptophan in the catalytic activity was confirmed by using DMHNB which is a specific tryptophan blocker. DMHNB (4 103 mol dm3) was found to inhibit the enzyme completely (data not shown).
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476
50 Gy 400 Gy
% Inhibition
100
50
0
-
Br2
(SCN)2Condition
I2-
Fig. 3. Radiation sensitivity of aminopeptidase in the presence of single electron oxidants. Purified aminopeptidase (20 mgm/ ml) was subjected to g-radiation in the presence of 1 103 mol dm3 KBr, KI and KSCN.
Fig. 4. Influence of gamma radiation on the absorption spectrum of aminopeptidase. A=Unirradiated. Before irradiation (400 Gy), the solutions were saturated with; B=Air; C=N2; D=N2O. Absorption at 280 nm was monitored.
3.4. Absorption and fluorescence characteristics The radiation-induced conformational changes associated with enzyme inactivation were monitored using optical absorption and fluorescence spectroscopy. The enzyme exhibited an absorption peak at 280 nm (Fig. 4) and a fluorescence emission at 340 nm (Fig. 5). The absorption at 280 nm which is attributed to aromatic amino acids, especially tyrosine (Lehninger et al., 1993), was found to increase on irradiation. This could be due to exposure of tyrosine residues from the core of the enzyme structure as a result of radiation-induced unfolding. Ionizing radiations are known to induce structural unfolding of proteins (Cie! sla et al., 2000; Tuce et al., 2001). However, it is interesting to note that the fluorescence peak at 340 nm, which is characteristic of tryptophan (Konev, 1967), decreased on irradiation (Fig. 5). The differential response of tyrosine and tryptophan residues to radiation could be explained on the basis of the enhanced sensitivity of the latter to ionizing radiations (Solar et al., 1984). The decrease in fluorescence at 340 nm (Fig. 5) is concomitant with the loss of enzyme activity (Fig. 1 and Table 1) suggesting strongly the contribution of tryptophan to the catalytic activity of aminopeptidase.
Fig. 5. Influence of g-radiation on the fluorescence emission of aminopeptidase. A=Unirradiated; B=50 Gy; C=400 Gy. Purified aminopeptidase was subjected to g-radiation and changes in fluorescence emission at 320 nm (lex ¼ 280 nm) were determined. Table 2 Rate constants (k in dm3 mol1 s1) for the reaction of aminopeptidase with some transients Transients
K
e aq
1 1010 1 1010 4 109 3 109 2 109
OH (SCN) 2 Br 2 I2
3.5. Pulse radiolysis studies The foregoing conclusions were based on steady-state radiation exposure where the observed effects would be a reflection of the cumulative and terminal contributions of long-lived as well as short-lived radiolytic species. In order to have a better understanding of transient species
formed during radiation inactivation, pulse radiolysis studies were carried out. Table 2 incorporates the rate constants for reaction of aminopeptidase with different radiolytic radicals. The enzyme reacts with OH and e aq with a rate constant of 1 1010 dm3 mol1 sec1. The
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0.006
477
0.010
0.010
a
320 nm
0.005
∆ O. D.
∆ O.D.
0.000
0.004
-0.005 -0.010
0
500
0.005
b
1000
0.002
0.000
0
AP / N2O
10
0.000 300
400
500
λ / nm Fig. 6. Transient absorption spectrum resulting from OH attack on aminopeptidase in solutions saturated with N2O. The spectrum is registered 2 ms after the pulse. Inset: Kinetic trace measured at 320 nm.
reaction rates of the enzyme with selected radicals were closest to those reported for pure tryptophan (Armstrong and Swallow, 1969; Cudina and Jovanovic, 1988). Solar et al. (1991) have shown that the reaction of Br with tryptophan leads to the formation of 2 , I2 tryptophan radical cation. Data on the transient signal generated by the interaction of OH radicals with aminopeptidase (Fig. 6), show that the presence of t-butanol markedly reduces the absorbance of the enzyme at 320 nm (Fig. 7). Thus the data from the pulse radiolysis studies strongly support the conclusions of our steady-state experiments, that tryptophan is an integral part of the catalytic site of chicken intestinal aminopeptidase. 3.6. Lifetime measurement
20
30
40
50
Time / µs
Fig. 7. Transient signal at 320 nm for reaction of aminopeptidase with OH radicals. In the absence (a) and presence (b) of t-butanol (2 102 mol dm3).
Table 3 Influence of radiation on the fluorescence lifetime of aminopeptidase components Component
Decay time (ns)
1 2 3
Unirradiated
Irradiated (400 Gy)
6.13 2.14 0.38
6.8 2.46 0.24
Table 4 Influence of radiation on the kinetics of aminopeptidase
3
Km (mmole/dm ) Vmax (mmoles/min) kcat kcat =Km (s/mmole/dm3)
Unirradiated
Irradiated (200 Gy)
111 1.01 1.8 0.162
500 0.357 0.66 0.0013
The time dependent fluorescence of the enzyme was determined using the single photon counting technique (Pal et al., 1990). The enzyme showed a threecomponent decay with lifetimes of i1 ¼ 6:13 ns, i2 ¼ 2:14 ns and i3 ¼ 0:38 ns. The decay components of the enzyme did not change significantly on irradiation (Table 3), indicating that there is little structural lesion to the protein molecule. This further affirms our contention that the radiation-induced loss of aminopeptidase activity is a direct consequence of active site modification leading to kinetic changes rather than structural lesion to the protein.
enzymes (Hasan et al., 1994; Abu El Faliat, 1983; Saha et al., 1992), gamma radiation brought about a change in the Km ; Vmax and the kcat of aminopeptidase. Irradiation at 200 Gy resulted in a three-fold decrease in the Vmax and a five-fold increase in Km ; providing evidence for significant impairement of the catalytic ability. The kcat value of the enzyme decreased to half following irradiation. These studies strongly indicate the predominant influence of radiation on the substratebinding site of the enzyme per se.
3.7. Influence of radiation on the enzyme kinetics
4. Conclusion
The results presented in Table 4 indeed demonstrate the changes in the enzyme kinetics consequent to radiation exposure. As observed in the case of other
Chicken intestinal aminopeptidase is inactivated by gamma radiation, predominantly due to the interaction of OH radicals. Data on the kinetics and spectral
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characteristics of the enzyme strongly point to alterations in the essential tryptophan and cysteine residues in the catalytic site as the main mechanism underlying the inactivation.
Acknowledgements The authors thank the Lady Tata Memorial Trust, Mumbai for the Junior Research Fellowship granted to Ms. V. K. Jamadar. Part of the data has been presented at the 3rd Asian Photochemistry Conference, 2002, Mumbai, India.
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Fielden, E.M., 1984. Chemical dosimetry of pulsed electron and X-ray sources in the 1–20 MeV range. In: Baxendale, J.H., Busi, F. (Eds.), The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis. Reidel, Dodrecht, pp. 49–62. Flores, M., Aristoy, M.C., Toldra, F., 1997. Curing agents affect aminopeptidase activity from porcine skeletal muscle. Z. Lebensm Unters Forsch A 205, 343–346. Fujimura, H., Ino, K., Nagasaka, T., Nakashima, N., Nakazato, H., Kikkawa, F., Mizutani, S., 2000. Aminopeptidase A expression in cervical neoplasia and its relationship to neoplastic transformation and progression. Oncology 58, 342–352. Guha, S.N., Moorthy, P.N., Kishore, K., Naik, D.B., Rao, K.N., 1987. One electron reduction of thrionine studied by pulse radiolysis. Proc. Indian Acad. Sci. (Chem. Sci) 99, 261–269. Hasan, N.M., Mc Call, P.R., Moore, J.S., Power, D.M., 1994. Radiation inactivation of bovine alkaline phosphatase. Radiat. Phys. Chem. 43, 233–237. Hui, M., Budari, E.D., Lajtha, A., Palkovitis, M., Hui, H.S., 1995. Changes in puromycin sensitive aminopeptidase in post mortem schizophrenic brain regions. Neurochem. Int. 27, 443–451. Jamadar, V.K., Jamdar, S.N., Dandekar, S.P., Harikumar, P., 2003. Purification and characterization of aminopeptidase from chicken intestine. J. Food Sci. 68, 438–443. Konev, S.V., 1967. Fluorescence and Phosphorescecne of Proteins and Nucleic Acids. Trans. Edition Sidney Udenfriend, Plenum Press, New York, pp. 61–105. Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993. Principles of Biochemistry. 2nd Edition. CBS Publ., pp. 109–133. Levin, C.H., Roselli, C.S., 2001. Radiation chemistry of proteins. In: Jonah, C.D., Rao, B.S.M. (Eds.), Radiation Chemistry—Present Status and Future Trends. Elsevier, Amsterdam, pp. 553–584. Masuda, T., Ovadia, J., Grossweiner, L.J., 1971. The pulse radiolysis and inactivation of trypsin. Int. J. Radiat. Biol. 20, 447–459. Mc Laughlin, W.L., Boyd, A.W., Chadwick, K.H., Mc Donald, J.C., Miller, A., 1989. In: Dosimetry for Radiation Processing. Taylor & Francis, London, pp. 144–147. Nagrani, S., Bisby, R.H., 1989. The radiation induced inactivation of external yeast invertase in dilute aqueous solution. Int. J. Radiat. Biol. 55, 191–200. Pal, H., Palit, D., Mukherjee, T., Mittal, J.P., 1990. Some aspects of steady state and time resolved fluorescence of tyrosine and related compounds. J. Photochem. Photobiol. A 52, 391–409. Preito, I., Martinez, A., Martinez, J.M., Ramirez, M.J., Vargas, F., Alba, F., Ramirez, M., 1998. Activities of aminopeptidase in rat saline model of volume hypertension. Horm. Metab. Res. 30, 246–248. Roberts, P.B., 1973. A radiation chemical study of the inactivation and active site composition of carboxypeptidase A. Int. J. Radiat. Biol. 24, 143–152. Saha, G.B., 2001. Physics and Radiation Biology of Nuclear Medicine. 2nd Edition. Springer, Berlin, pp. 181–212. Saha, A., Mandal, P.C., Bhattacharrya, S.N., 1991. Radiation induced changes in flavin fluorescence of dihydroorotate dehydrogenase. Int. J. Radiat. Biol. 60, 769–778.
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Radiation Physics and Chemistry 69 (2004) 473–479
Radiation inactivation study of aminopeptidase: probing the active site V.K. Jamadara, S.N. Jamdara, Hari Mohanb, S.P. Dandekarc, P. Harikumara,* a
Food Technology Division, Flesh Food Biochemistry Section, BARC, Mumbai 400 085, India b Radiation Chemistry & Chemical Dynamics Division, BARC, Mumbai 400 085, India c Department of Biochemistry, Seth G. S. Medical College, KEM Hospital, Mumbai 400 012, India Received 8 April 2003; accepted 30 September 2003
Abstract Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N2O>N2>air. The D37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N2O. The radicals (SCN) 2 , Br 2 and I2 inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiationinduced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies. r 2003 Elsevier Ltd. All rights reserved. Keywords: Radiation inactivation; Aminopeptidase; Pulse radiolysis; Free radicals
1. Introduction Radiation-induced free radicals interact strongly with biomolecules such as DNA (Saha, 2001), proteins (Levin and Roselli, 2001), lipids and other membrane components (Stark, 1991). Enzymes being the core functional units, their inactivation bears a significant impact on the cell metabolism (Saha et al., 1995). Hence, the radiation chemistry of proteins and enzymes assumes paramount importance. From the radiation biology point of view, the free radical chemistry of enzymes has a longstanding interest since such studies give information about the properties of the active site and migration of free radicals through macromolecules (von Sonntag, 1987). The radiation-induced effects have been carried out with major emphasis on the enzymes involved in oxidation– *Corresponding author. Tel.: +91-99224404949; fax: +91225505151. E-mail address: [email protected] (P. Harikumar).
reduction reactions (Saha et al., 1991; Buchanan and Armstrong, 1976, 1978; Abu El Faliat et al., 1983). Among proteases, papain (Clement et al., 1974), trypsin (Masuda et al., 1971; Cudina and Jovanovic, 1988) and carboxypeptidase (Roberts, 1973) have been well studied for their response to ionizing radiation. However, very few efforts have been directed towards understanding the radiation chemistry of N terminal cleaving exopeptidases viz. aminopeptidases. Aminopeptidases (E.C. 3.4.11. ) are a major class of exopeptidases implicated in several pathological conditions (Hui et al., 1995; Preito et al., 1998; Fujimura et al., 2000; Tomoharu et al., 2001) and in general cellular homeostasis (Taylor, 1993). This class of enzymes also finds extensive application in the food industries related to cheese ripening, dairy products and preparation and processing of protein hydrolysates (Flores et al., 1997; Bouchier et al., 2001). Information on the structure–function relationship of aminopeptidases has not been easily forthcoming due to their lability and multimeric
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ARTICLE IN PRESS V.K. Jamadar et al. / Radiation Physics and Chemistry 69 (2004) 473–479
structural features (Taylor, 1993). In the present study, an attempt has been made to evaluate the influence of gamma radiation and pulse radiolysis as a tool to probe into the active site composition of an aminopeptidase purified to homogeneity (Jamadar et al., 2003) from chicken intestine. Chicken intestine is an important byproduct of the poultry industry, currently being exploited as a rich source of proteins and proteolytic enzymes in our laboratory.
2. Experimental l-arginine-b-naphthylamide (ANA) and mersalyl acid were obtained from Sigma Co., USA. All other chemicals were of Analar grade. N2 and N2O gases used were of IOLAR grade (Indian Oxygen Ltd.) having a purity of X99.9%. The activity of aminopeptidase was determined by the method of Barrett and Kirchke (1981). The assay was carried out at 50 C in 0.1 mol dm3 phosphate buffer pH 6.0 containing 1 103 mol dm3 cysteine using 2 102 mol dm3 ANA as a substrate. The reaction was terminated by addition of 1 103 mol dm3 mersalyl reagent and the pink colour complex was read at 520 nm. Enzyme kinetics including Km ; Vmax and kcat were determined before and after irradiation using the Lineweaver– Burke’s Plot. Aqueous solutions of aminopeptidase (5 103 mol dm3) in 0.25 mol dm3 NaCl and 1 102 mol dm3 phosphate buffer pH 7.0 were irradiated using a Gamma cell 220. The dose rate was 8.5 Gy min1 as determined by Fricke dosimetry (McLaughlin et al., 1989). A 7 MeV linear accelerator (Forward Industries, UK) with a single pulse of 50 ns was used for pulse radiolysis experiments. The detailed working of the system was as described by Guha et al. (1987). The dose rate was determined according to Fielden (1984). For studying the OH radical reactions, the enzyme solution was flushed with N2O gas (IOLAR grade, purity X 99.9%.), to convert all the e aq to OH radicals (Buxton, 1987). The absorption and fluorescence spectra were recorded using the Hitachi 330 spectrometer and Hitachi F 4010 spectrofluorimeter, respectively. The fluorescence lifetime of the enzyme was determined on an EI 199 fluorescence spectrometer (Edinburg, U.K) using the time-correlated single photon counting technique (Pal et al., 1990). The D37 value of the enzyme was determined as the dose required to bring about 63% inactivation of the enzyme. G value was calculated as the number of enzyme molecules inactivated per 100 eV of energy absorbed, and was reported in micromoles/joule (dose rate=8.5 Gy min1) from the slope of the graph plotting D37 vs enzyme concentration (Sanner and Pihl,
1969). The values of percentage inactivation of enzyme are in the range of78–10%.
3. Results and discussion 3.1. Influence of graded dose and irradiation conditions The influence of absorbed radiation dose and conditions of irradiation on aminopeptidase is presented in Fig. 1 and Table 1, respectively. As reported for other enzymes (Sui, 1973; Roberts, 1973), the radiation inactivation of aminopeptidase exhibited an exponential relationship with radiation dose. The exponential relationship observed for aminopeptidase could be due to the non discriminatory interaction of radiolytic free radicals with the active and inactivated enzyme molecules, as pointed out by Sanner and Pihl (1969), for enzymes in general. Data on the influence of different gases in the irradiation media (Table 1) showed that the inactivation was in the order N2O>N2>air with D37 values of 198, 210 and 281 Gy, respectively, indicating thereby that the enzyme inactivation is predominantly OH mediated. The relatively lower effect in the aerated medium as compared to anaerobic conditions could be
10 0
% Residual activity
474
80
60
40
20
0
100
200
300
400
Dose (Gy)
Fig. 1. Effect of absorbed radiation dose on aminopeptidase. Purified aminopeptidase (17 mg/ml) in 0.25 mol dm3 NaCl in 1 102 mol dm3 phosphate buffer pH 7.0 was subjected to g-radiation. The residual enzyme activity was determined under standard assay conditions.
Table 1 D37 and G values of aminopeptidase
D37 (Gy) G value (mmoles/Joule)
Air
N2
N2O
281 0.011
210 0.018
198 0.024
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d
H
þO2 -HOd2 ;
d e aq þO2 -O2 ;
10
1 1
3
k ¼ 2 10 dm mol 10
3
k ¼ 1:9 10 dm mol
s ;
1 1
s ;
ð1Þ ð2Þ
Absence of OH scavenger Isopropanol t-Butanol
120
100
Residual Activity (%)
ascribed, as suggested by Spinks and Woods (1976), to the scavenging of e aq and H atoms by dissolved oxygen, which results in the formation of superoxide anion (O 2 ) and hydroperoxyl radical (HO2 ), respectively (reactions (1)–(3)). These radicals react much slowly compared toOH.
475
80
60
40
HOd2 ¼ Hþ þOd 2 ;
pK ¼ 4:8:
ð3Þ
The G value of aminopeptidase in media saturated with N2O was double that observed in air because e aq is converted to OH radicals (reaction (4)). d e aq þN2 O- OH þ OH þN2 ;
k ¼ 0:91 1010 dm3 mol1 s1 ;
ð4Þ
This is in agreement with the observations of Buchanan and Armstrong (1976) for lactate dehydrogenase. As reported by Winchester and Lynn (1970) for the dipeptide try–gly (Go0:1), G value between 0.01 and 0.03 observed for aminopeptidase (Table 1) could be due to the conversion of tryptophan residues in the enzyme to 3-OH kynurenin or DOPA. However, aminopeptidase being a multimeric polypeptide, the contribution by other residues cannot be ruled out. 3.2. Effect of OH scavengers The role of OH radicals in the inactivation of aminopeptidase was confirmed by studies employing t-butanol and isopropanol which are reported to be scavengers of OH (Nagrani and Bisby, 1989; Adams et al., 1969). Isopropanol scavenges both H, and OH (reactions (5) and (6)). ðCH3 Þ2 CHOH þd OH-H2 O þ ðCH3 Þ2 Cd OH; k ¼ 2 1010 dm3 mol1 s1 :
ð5Þ
ðCH3 Þ2 CHOH þd H-H2 þðCH3 Þ2 C OH; k ¼ 1 108 dm3 mol1 s1 :
ð6Þ
Isopropanol and t-butanol elicited 80% and 95% protection, respectively, to aminopeptidase against radiation-induced inactivation (Fig. 2). This observation reaffirms our contention that the radiation inactivation of this enzyme is predominantly caused by OH radicals. In the case of t-butanol, although H atoms may be interacting with the enzyme, it apparently does not lead to a change in enzyme activity. 3.3. Effect of single electron oxidants on aminopeptidase Radiolytic radicals such as H, OH and e aq could react at random on several sites of a protein but all
20
0
100
200
300
400
Dose (Gy)
Fig. 2. Radiation sensitivity of aminopeptidase in the presence of OH scavengers. Purified aminopeptidase was subjected to gamma radiation in the presence of 1 103 mol dm3 t-butanol and isopropanol. The enzyme solution was purged with N2O gas before irradiation.
reactions need not lead to inactivation. Hence, changes in the enzyme activity per se by these radicals do not point to the specific sites of interactions (Bisby et al., 1974). Hence it would be interesting to use single electron oxidants (viz. Br and (SCN) 2 , I2 2 ) and biochemical modifiers (DMHNB-[Dimethyl (2-hydroxy5-nitrobenzyl) sulfonium bromide]) to gain a better insight into the radiation-sensitive residues in the enzyme. These inorganic radicals are known to react with specific amino acids (Adams et al., 1972). At neutral pH, Br reacts with tyrosine, tryptophan, 2 histidine and cysteine, (SCN) 2 reacts with tryptophan, tyrosine, cysteine and methionine while I reacts only 2 with cysteine (Hasan et al., 1994). These chemical probes can thus be used to identify the radiation-sensitive residues in the enzyme which may be crucial to its catalytic function. The data presented in (Fig. 3) show that gamma radiation (400 Gy) inactivated aminopepti dase by 96%, 85% and 80% in I 2 , (SCN)2 and Br2 , respectively. The observation that the enzyme was completely inactivated in the presence of the above radicals suggests the involvement of aromatic and thiol amino acids in the catalytic activity of the enzyme. The highest effectiveness of I 2 points to the participation of cysteine residues. In fact our earlier studies using cysteine, 2-mercaptoethanol and dithiothreitol have shown that the number of available SH residues is a critical factor in eliciting the activity of the enzyme (Jamadar et al., 2003). The decisive role of tryptophan in the catalytic activity was confirmed by using DMHNB which is a specific tryptophan blocker. DMHNB (4 103 mol dm3) was found to inhibit the enzyme completely (data not shown).
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476
50 Gy 400 Gy
% Inhibition
100
50
0
-
Br2
(SCN)2Condition
I2-
Fig. 3. Radiation sensitivity of aminopeptidase in the presence of single electron oxidants. Purified aminopeptidase (20 mgm/ ml) was subjected to g-radiation in the presence of 1 103 mol dm3 KBr, KI and KSCN.
Fig. 4. Influence of gamma radiation on the absorption spectrum of aminopeptidase. A=Unirradiated. Before irradiation (400 Gy), the solutions were saturated with; B=Air; C=N2; D=N2O. Absorption at 280 nm was monitored.
3.4. Absorption and fluorescence characteristics The radiation-induced conformational changes associated with enzyme inactivation were monitored using optical absorption and fluorescence spectroscopy. The enzyme exhibited an absorption peak at 280 nm (Fig. 4) and a fluorescence emission at 340 nm (Fig. 5). The absorption at 280 nm which is attributed to aromatic amino acids, especially tyrosine (Lehninger et al., 1993), was found to increase on irradiation. This could be due to exposure of tyrosine residues from the core of the enzyme structure as a result of radiation-induced unfolding. Ionizing radiations are known to induce structural unfolding of proteins (Cie! sla et al., 2000; Tuce et al., 2001). However, it is interesting to note that the fluorescence peak at 340 nm, which is characteristic of tryptophan (Konev, 1967), decreased on irradiation (Fig. 5). The differential response of tyrosine and tryptophan residues to radiation could be explained on the basis of the enhanced sensitivity of the latter to ionizing radiations (Solar et al., 1984). The decrease in fluorescence at 340 nm (Fig. 5) is concomitant with the loss of enzyme activity (Fig. 1 and Table 1) suggesting strongly the contribution of tryptophan to the catalytic activity of aminopeptidase.
Fig. 5. Influence of g-radiation on the fluorescence emission of aminopeptidase. A=Unirradiated; B=50 Gy; C=400 Gy. Purified aminopeptidase was subjected to g-radiation and changes in fluorescence emission at 320 nm (lex ¼ 280 nm) were determined. Table 2 Rate constants (k in dm3 mol1 s1) for the reaction of aminopeptidase with some transients Transients
K
e aq
1 1010 1 1010 4 109 3 109 2 109
OH (SCN) 2 Br 2 I2
3.5. Pulse radiolysis studies The foregoing conclusions were based on steady-state radiation exposure where the observed effects would be a reflection of the cumulative and terminal contributions of long-lived as well as short-lived radiolytic species. In order to have a better understanding of transient species
formed during radiation inactivation, pulse radiolysis studies were carried out. Table 2 incorporates the rate constants for reaction of aminopeptidase with different radiolytic radicals. The enzyme reacts with OH and e aq with a rate constant of 1 1010 dm3 mol1 sec1. The
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0.006
477
0.010
0.010
a
320 nm
0.005
∆ O. D.
∆ O.D.
0.000
0.004
-0.005 -0.010
0
500
0.005
b
1000
0.002
0.000
0
AP / N2O
10
0.000 300
400
500
λ / nm Fig. 6. Transient absorption spectrum resulting from OH attack on aminopeptidase in solutions saturated with N2O. The spectrum is registered 2 ms after the pulse. Inset: Kinetic trace measured at 320 nm.
reaction rates of the enzyme with selected radicals were closest to those reported for pure tryptophan (Armstrong and Swallow, 1969; Cudina and Jovanovic, 1988). Solar et al. (1991) have shown that the reaction of Br with tryptophan leads to the formation of 2 , I2 tryptophan radical cation. Data on the transient signal generated by the interaction of OH radicals with aminopeptidase (Fig. 6), show that the presence of t-butanol markedly reduces the absorbance of the enzyme at 320 nm (Fig. 7). Thus the data from the pulse radiolysis studies strongly support the conclusions of our steady-state experiments, that tryptophan is an integral part of the catalytic site of chicken intestinal aminopeptidase. 3.6. Lifetime measurement
20
30
40
50
Time / µs
Fig. 7. Transient signal at 320 nm for reaction of aminopeptidase with OH radicals. In the absence (a) and presence (b) of t-butanol (2 102 mol dm3).
Table 3 Influence of radiation on the fluorescence lifetime of aminopeptidase components Component
Decay time (ns)
1 2 3
Unirradiated
Irradiated (400 Gy)
6.13 2.14 0.38
6.8 2.46 0.24
Table 4 Influence of radiation on the kinetics of aminopeptidase
3
Km (mmole/dm ) Vmax (mmoles/min) kcat kcat =Km (s/mmole/dm3)
Unirradiated
Irradiated (200 Gy)
111 1.01 1.8 0.162
500 0.357 0.66 0.0013
The time dependent fluorescence of the enzyme was determined using the single photon counting technique (Pal et al., 1990). The enzyme showed a threecomponent decay with lifetimes of i1 ¼ 6:13 ns, i2 ¼ 2:14 ns and i3 ¼ 0:38 ns. The decay components of the enzyme did not change significantly on irradiation (Table 3), indicating that there is little structural lesion to the protein molecule. This further affirms our contention that the radiation-induced loss of aminopeptidase activity is a direct consequence of active site modification leading to kinetic changes rather than structural lesion to the protein.
enzymes (Hasan et al., 1994; Abu El Faliat, 1983; Saha et al., 1992), gamma radiation brought about a change in the Km ; Vmax and the kcat of aminopeptidase. Irradiation at 200 Gy resulted in a three-fold decrease in the Vmax and a five-fold increase in Km ; providing evidence for significant impairement of the catalytic ability. The kcat value of the enzyme decreased to half following irradiation. These studies strongly indicate the predominant influence of radiation on the substratebinding site of the enzyme per se.
3.7. Influence of radiation on the enzyme kinetics
4. Conclusion
The results presented in Table 4 indeed demonstrate the changes in the enzyme kinetics consequent to radiation exposure. As observed in the case of other
Chicken intestinal aminopeptidase is inactivated by gamma radiation, predominantly due to the interaction of OH radicals. Data on the kinetics and spectral
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characteristics of the enzyme strongly point to alterations in the essential tryptophan and cysteine residues in the catalytic site as the main mechanism underlying the inactivation.
Acknowledgements The authors thank the Lady Tata Memorial Trust, Mumbai for the Junior Research Fellowship granted to Ms. V. K. Jamadar. Part of the data has been presented at the 3rd Asian Photochemistry Conference, 2002, Mumbai, India.
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