Immobilization of Adenosine Deaminase onto Agarose and Casein

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Biomacromolecules 2002, 3, 432-437

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Immobilization of Adenosine Deaminase onto Agarose and Casein A. M. Dessouki*,† and K. S. Atia‡ National Center for Radiation Research and Technology, P.O. Box 29, Nasr City, Cairo 11731, Egypt, and Nuclear Research Center, Inshas, Egypt Received August 21, 2001; Revised Manuscript Received January 6, 2002

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of γ-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of γ radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to γ radiation up to a dose of 5 Mrad. Introduction Adenosine deaminase (ADA) is classified as a hydrolase, acting on the carbon-nitrogen bond other than peptide bonds in cyclic amidines. The highest activity of ADA was found in animal small intestine mucosa, appendix, and spleen, localized cell cytoplasm and nucleus.1 ADA had many biological and medical applications, it was used in the determination of 5′-nucleotidase in blood and in the synthesis of inosine, deoxyinosine, and dideoxyinosine. It is used also in deamination of adenosine analogues to corresponding inosine analogues. The primary function of ADA is the detoxification of pharmacologically active adenosine. ADA deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. An improved form of treatment was developed by covalent attachment of poly(ethylene glycol) (PEG) to the purified bovine enzyme. The main biochemical consequences of ADA deficiency are almost completely reversed by PEG-ADA treatment, resulting in an increase in circulating T lymphocytes and improved of cellular immune functions.2 The immobilization of ADA onto different carriers was studied by several authors who reported some biological and medical application of the immobilized ADA. They used the immobilized technique for the construction of an enzyme electrode for adenosine.3-6 Carriers based on natural polymers of the polysaccharide types (such as cellulose, dextran, starch, chitin, or agarose) can be useful alternatives to inorganic material due to their * To whom correspondence may be addressed. † National Center for Radiation Research and Technology. ‡ Nuclear Research Center.

well-defined pore size. Casein, the major protein of bovine milk, is heterogeneous consisting of R-, β-, and γ-casein (75%, 22%, and 3%, respectively) and is heat-resistant as well as acid labile protein. Experimental Section Materials. The enzyme adenosine deaminase (ADA) (EC 3.5.4.4, type IV; from bovine spleen), adenosine, epichlorohydrine, and agarose were purchased from Sigma Chemical Co., St. Louis, MO (USA). Casein was obtained from EL-Nasr Chemical Co., Abo-Zable (Egypt), Glutaraldehyde (GA) (purum approximately 25% water) was from Ferak, Berlin (Germany), and monobasic sodium phosphate and dibasic sodium phosphate were purchased from Merck (Germany). Other chemicals were of analytical grade. Techniques. A 60Co γ-ray source with a dose rate of 1.22 kG/h was used as the γ-ray source. Enzyme Assay. The activity of ADA was measured by a spectrophotometric method (using Miltron Roy 1201 spectrophotometer) based on that of Solmon.7 The optical density of 3 mL of adenosine solution (containing 20 mg of adenosine per mL of 0.1 M phosphate buffer pH 7.5) was measured at 265 nm immediately after addition of native ADA (44 mg of protein) and again after incubation at 37 °C for 1 h, the spectrophotometer being at zero, using 44 mg of protein in 3 mL of 0.1 M phosphate buffer pH 7.5 as a blank which was incubated in parallel with a test solution. For assaying of immobilized enzyme activity, an appropiate weight of biopolymer (enzyme and carrier), containing approximately 44 mg of protein, was measured at 265 nm

10.1021/bm0156025 CCC: $22.00 © 2002 American Chemical Society Published on Web 04/26/2002

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Immobilization of Adenosine Deaminase

immediately and again after incubation at 37 °C for 1 h in a shaker water bath. The fall of the optical density after the above conditions was multiplied by the factor 123 to give the international units (1 µmol of adenosine deaminatedmin-1L-1).7 Protein Determination. The concentration of immobilized ADA was determined from the difference between the amount of protein introduced to the coupling reaction mixture and the amount of protein present in the filtrate and washings after immobilization. The amount of protein was determined by the Lowry method.8,9 Binding of the Linker to the Carrier. (a) Binding of Epichlorohydrin to Casein To Give C-Epi. Five grams of casein and 0.6 g of NaOH in 50 mL of distilled water were stirred at 30 °C for 24 h, after which, 1 mL of epichlorohydrin was added dropwise, and the reaction mixture was stirred for 6 h at 25 °C. The treated casein was mixed with 25 mL of ethanol, the mixture was filtered and washed with 3 × 10 mL of distilled water, and water was replaced from the gel by adding 25 mL of ethanol. Finally, ethanol was evaporated at 40 °C, and the activated carrier was dried and stored at 4 °C. (Yield: 73%.) (b) Binding of Epichlorohydrin to Agarose To Give (AEpi). Twenty grams of agarose in 100 mL of methanol and 1.2 g of NaOH were stirred at 30 °C for 24 h, after which, a mixture of 25 mL of 95% ethanol and 10 mL of epichlorohydrin containing 2.5 g of NaOH were add dropwise, and the reaction mixture was stirred for 6 h at 30 °C. The activated agarose was filtered and washed with 3 × 10 mL distilled water, and the activated carrier was dried and stored at 4 °C. (Yield: 85%.) Preparation of Immobilized Enzymes. (a) Immobilization of ADA on Activated Casein (C-Epi). Two milliliters of enzyme solution (0.87 mg of protein; i.e., 42 IU in 2 mL of 0.1 M phosphate buffer, pH 7.5) was added to 1 g of activated casein in 45 mL of 0.1 M phosphate buffer, pH 7.5, and the mixture was shaken for 24 h at room temperature. After reaction, the unbound enzyme fraction was removed by washing with 3 × 3 mL of phosphate buffer, pH 7.5, and then by 2 × 5 mL of distilled water. After the last addition of water, the gel was exhaustively vacuum filtered to remove water between the particles. (Yield: 32.6%.) (b) Immobilization of ADA on Casein with Glutaric Dialdehyde (Pentane-1,5-dial). Two mililiters of enzyme solution (0.87 mg of protein; i.e., 42 IU in 2 mL of 0.1 M phosphate buffer, pH 7.5) was added to 1 g of casein (previously shaken for 2 h at 25 °C in 5 mL of glutaraldehyde solution (25% w/w) and 45 mL of 0.1 M phosphate buffer, pH 7.5), and the mixture was shaken for 24 h at room temperature. The unbound enzyme fraction was removed by washing as mentioned above. (Yield: 86.2%.) (c) Immobilization of ADA on Activated Agarose (AEpi). Two milliliters of enzyme solution (0.87 mg of protein; i.e., 42 IU in 2 mL of 0.1 M phosphate buffer, pH 7.5) was added to 1 g of activated agarose in 45 mL of 0.1 M phosphate buffer, pH 7.5, and the mixture was shaken for 20 h at room temperature. After this time, the gel was washed thoroughly by adding several volumes of distilled water and

Table 1. Effect of pH Incubation Time on ADA Linked to Activated Agarose and Casein protein immobilized, mg/g of support

relative activity (%)

pH

A-Epi

C-Epi

A-Epi

C-Epi

6 6.5 7 7.5 8

0.652 0.661 0.722 0.791 0.678

0.578 0.617 0.678 0.748 0.635

73.8 76.2 80.9 90.4 76.2

64.3 69.0 76.2 85.6 71.4

vacuum filtered after each addition. After the last addition of water, the gel was exhaustively vacuum filtered to remove water between the particles. (Yield: 28%.) (d) Immobilization of ADA on Agarose with Glutaraldehyde (GA). One gram of agarose was shaken for 2 h at 25 °C in 5 mL of glutaralehyde solution (25% w/w) and 45 mL of 0.1 M phosphate buffer, pH 7.5, then 2 mL of enzyme solution (0.87 mg of protein; i.e., 42 IU in 2 mL of 0.1 M phosphate buffer, pH 7.5) was added to 1 g of agarose and the mixture was shaken for 24 h at room temperature. After the reaction, the unbound enzyme fraction was removed by washing as mentioned above. (Yield: 91.5.) Establishment of ADA Immobilization. (a) Effect of Incubation pH. The activated supports were incubated for 24 h at 4 °C with 42 units of ADA activity in 25 mL of 0.1 M sodium phosphate buffer at different pH values ranging from 6 to 8. The maximum activity of immobilized ADA was found at pH 7.5. (b) Effect of ADA Concentration. Different ADA concentrations ranging from 2 to 60 units (0.52 to 1.52 mg of protein) were added to 1 g of activated support in sodium phosphate buffer, pH 7.5, at 4 °C for an incubation time of 8 h. The maximum coupling of ADA was achieved at the concentration of 42 U/g. Results and Discussion Establishment of ADA Immobilization. (a) Factors Affecting the Amount of Enzyme Attachment. (i) Effect of Incubation pH. Table 1 shows the effect of changing incubation pH and the precentage of maximum activity of immobilized ADA which was found to increase from 64.3% at pH 6 to 85.6% at pH 7.5 and from 73.7% at pH 6 to 90.4% at pH 7.5 for agarose-ecpichlorohydrin and caseinepichlorohydrin, respectively. The immobilization reaction by an epichlorohydrin-activated agarose is based on the alkylation of the amino, phenolic, and thiol groups of enzyme. This reaction is nucleophilic in nature; therefore the alkaline pH favors the reaction,7 but at elevated pH the enzyme is deactivated. The opening of the oxide ring in this reaction represents a nucleophilic displacement on carbon of the oxide to give the intermediate II which, by acquiring a proton, gives the open product III.

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Figure 1. Effect of incubation time on linking ADA to agaroseepichlorohydrine and casein-epichlororhydrine. Forty nine units of ADA enzyme was applied on 1 g of matrix suspended on 0.1 M sodium phosphate buffer pH 7.5, incubated for 16 h at 4 °C.

It was reported that the rate of inactivation of enzymes, like other protein denaturations, is in most cases greatly dependent on the pH of the solution. The protonation and deprotonation of the charged functional groups are dependent upon the pH of the solution. The increase and decrease in hydrogen ion concentration will diminish electrostatic attraction as the balance of charge is distributed leading to the disruption of intermolecular hydrogen bonding. The decrease or increase in overall positive and negative charges provides enough repulsion force to overcome the nonpolar cohesion between the smaller molecular weight units. This applies to our results and is in good agreement with several authors.7,11,12 (ii) Effect of ADA Concentration. The maximum amount of ADA was achieved at concentration 42 units. A slight increase in immobilization percent was detected with increasing the enzyme concentration up to 0.87 mg of protein. This was followed by a decrease to the original retention activity at 1.25 mg of protein. However, increasing the amount of added protein resulted in a decrease in retention activity.12 (iii) Effect of Incubation Time. ADA was incubated with activated agarose and activated casein at pH 7.5 and at 4 °C. The amount of ADA attached increased fast up to an incubation time of about 6 h, beyond which it increased gradually and, finally, remained constant after 8 h (Figure 1). At the optimum pH (pH 7.5), the ADA required 8 h (optimum incubation time) at 4 °C to completely saturate all active sites on the activated agarose and activated casein. Beyond the optimum incubation time, there was no change in the activity of the immobilized ADA. This is because of exhaustion of the active sites, so the enzyme may not have sites on the support. This result was nearly similar to the optimum incubation time of immobilized R-amylase and glucoamylase on cyanuric chloride cellulose ether at 4 °C, where they required 12 and 18 h for maximum immobilization.13 Relative activities of 31% and 24% were obtained for covalent immobilized ADA and glutaraldehyde immobilized ADA, respectively. Physcio-Chemical properties of free and immobilized ADA. (a) Operational Stability. To evaluate the usability

Dessouki and Atia

Figure 2. Reusability of immobilized ADA preparations (stored at 4 °C). Enzyme activity was determined at 25 °C in 0.1 M sodium phosphate buffer, pH 7.5. The preparations of ADA investigated were (AGA) immobilized on agarose-glutaraldehyde, (A-Epi) immobilized on agarose-epichlorohydrin, (C-GA) immobilized on casein-glutaraldehyde, and (C-Epi) immobilized on casein-epichlorohydrin.

of immobilized ADA, the enzyme immobilized in agarose using glutaric dialdehyde (GA) as cross-linker, agaroseepichlorohydrin, casein using glutaric dialdehyde (GA) and casein-epichlorohydrin, a series of experiments in the batch system were carried out. The activities of the first batch were taken to be 100%.14 Figure 2 shows the relative activity as a function of reuse number for the four cases studied. It can be seen from the results that the reuse of the immobilized enzymes was up to 10 times. Agarose-GA and casein-GA resulted in a sharp decrease in the relative activity 36% and 25%, respectively, while agarose-epichlorohydrin and caseinepichlorohydrin showed only slight decrease in relative activity after 10 times, 88% and 84%, respectively. This high stability suggests that very little leakage of the immobilized enzymes (agarose-epichlorohydrin and casein-epichlorohydrin) occurred under repeated washing. This high stability is in marked contrast with the rather poor durability of the agarose-GA and casein-GA. Similar conclusions were reported by several working with different enzymes, polymers, and cross-linkers.14 (b) Temperature Optimum. The activity of free and immobilized ADA was assayed at various temperatures (1040 °C) in a thermostatically controlled water bath under standard assay conditions. The results in Figure 3 showed the temperature optima of immobilized ADA shifted slightly toward higher temperature from 25 to 30 °C. A similar increase in temperature optima had been found in immobilized β-amylase15 and R-amylase,16 but a reverse effect was shown by immobilized urease.17 The increase in temperature optima for the activity of immobilized enzyme could be due to the fact that the actual temperature in the microenvironment of the matrix was lower than that in bulk solution.18 It is well-known that the activity of immobilized enzymes, especially in a covalently bound system, is more resistant against heat and denaturing agents than the soluble form. (c) Thermal Inactivation. The thermoinactivation kinetics at 40 and 50 °C of the soluble and immobilized enzyme showed remarkable achievement of thermostability by the immobilized form (Figure 4) with enhanced half-life. This thermostability was higher at 40 °C than at 50 °C under the

Immobilization of Adenosine Deaminase

Figure 3. Effect of temperature on the activity of both free and immobilized ADA. Appropriate quantities of soluble and immobilized ADA preparations were assayed at the indicated temperature under standard assay conditions.

Figure 4. Thermoinactivation kinetics of both free and immobilized ADA exposed to 40 °C. Appropriate aliquots of soluble and immobilized preparations were incubated at 40 °C in 0.1 M sodium phosphate buffer, pH 7.5, for the indicated durations and chilled quickly for 15 min, and enzyme activities were determined under standard assay conditions.

same conditions. This increased tolerance to thermal denaturation may be due to multipoint attachment of ADA molecules to the polymer carrier through reduction in molecular mobility.19 (d) pH Optima and Stability. The behavior of an enzyme molecule may be modified by its immediate microenvironment. An enzyme in solution can have a different pH optimum from the same enzyme immobilized on a solid matrix depending on the surface and residual charges on the solid matrix and the nature of the enzyme-bound pH value in the immediate vicinity of the enzyme activity. A change in the optimum pH normally accompanies the insolubilization of enzymes, depending upon the polymer used as support. Since the enzyme activity is markedly influenced by environmental conditions, especially pH activity, the behavior caused by enzyme immobilization is useful for understanding the structure-function relationships of enzyme proteins.

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Figure 5. pH stability of free and immobilized ADA. Soluble and immobilized ADA preparations were incubated at 25 °C at various pH values (5-10) for 30 min in phosphate buffer, 0.1 M, and the activity was measured under standard assay conditions.

Figure 6. Storage stability at 4 °C of the free and immobilized ADA (dry state). Enzymes activity was determined at 25 °C in sodium phosphate buffer pH 7.5.

Therefore, it is very useful to compare the activity of soluble and immobilized enzyme as a function of pH.19 The activity of the free ADA was compared with that of immobilized preparation at various pH values (6-10) in phosphate buffer at pH 7.5, and the activity was measured under the standard assay conditions. The immobilized adenosine deaminase has the same pH optima as the free one, but the pH profile is considerably widened due to diffusional limitations.21-23 Soluble and immobilized ADA were kept at 25 °C at various pH values (5-10) for 30 min in phosphate buffer, 0.1 M, and the activity was measured under the standard assay conditions, and it showed that the immobilized enzyme has more stability than the free enzyme (Figure 5). (e) Storage Stability. Figure 6 shows the relative activity of ADA stored at 4 °C as a function of time for the free ADA and immobilized ADA onto both activated agarose and

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Figure 7. Storage stability at 25 °C of the free and immobilized ADA (dry state). Enzymes activity was determined at 25 °C in sodium phosphate buffer pH 7.5.

Figure 8. Storage stability at 25 °C of the free and immobilized ADA (wet state). Enzymes activity was determined at 25 °C in sodium phosphate buffer pH 7.5.

activated casein stored in the dry state (in phosphate buffer, pH 7.5). It can be seen from the results that the relative activity of the free enzyme dropped from 100% to 20% after a storage time of 160 days. However, the drop in relative activity for the dry state of the immobilized enzyme was remarkable compared to that of the wet state. The higher stability of the immobilized ADA can be attributed to preventation of autodigestion and thermal denaturation as a result of the fixation of ADA molecules on the surface of the activated agarose and activated casein. However, it is often pointed out that lyophilization of enzymes directly from the water suspensions is normally accompanied by loss of the enzymatic activity. This is in good agreement with our results. Figures 7 and 8 show the storage of ADA at 25 °C under the same conditions as above. It was observed that the relative activity decreased markedly with time. After 30 days of storage, the relative activity of the free enzyme dropped to 35%. As expected, the loss in relative activities when

Dessouki and Atia

Figure 9. Effect of γ radiation on the activity of free and immobilized ADA.

storing at 25 °C is much greater than when the enzyme was stored at 4 °C.14 (f) Effect of γ Radiation. The effect of γ radiation on the free and the immobilized ADA was investigated, and the results are shown in Figure 9. The free ADA lost about 50% from its relative activity when exposed to a dose of 1 Mrad and lost the rest of its activity at a dose of about 2 Mrad. However, when ADA was immobilized onto activated agarose and activated casein, it showed more radiation resistance.24,25 The activity of immobilized ADA, irradiated to a dose of 4 Mrad, showed no significant change in its relative activity, while a reduction of 50% was measured at a dose of 10 Mrad. It is obvious that the immobilization of the enzyme onto polymeric support resulted in good radiation resistance of the enzyme. It seems that the polymer stabilized the produced free radicals and protected the enzyme. Also, the immobilization of ADA onto the polymers and the subsequent exposure to γ radiation resulted in the sterilization of the enzyme and hence the possibility of using the sterilized immobilized enzyme in different applications such as in the manufacturing of drugs and also in other applications.26-28 Kinetic Properties. Reaction kinetics was analyzed for free and immobilized ADA in phosphate buffer, pH 7.5, at 37 °C by using different concentrations of adenosine (0.0060.05 mol/L). Calculations were done according to the equation 1/V ) 1/Vmax + Km/Vmax 1/[S] where Km is the Michaelis-Menten constant (mmol/dm-3) and Vmax is the maximum initial rate in the MichaelisMenten equation (mmol dm-3 min-1). The data in Table 2 show the kinetic parameters of both free and immobilized ADA. The observed differences between free and immobilized ADA kinetics can be explained in terms of the structural changes caused by binding of the enzyme onto different supports. For agaroseepichlorohydrine (A-Ei) amd casein-epichlorohydrin (CEpi), the increase of Km and decrease of Vmax values is as expected because of the blocking of active sites of the

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Immobilization of Adenosine Deaminase Table 2. Kinetic Parameters of Free and Immobilized ADA enzyme form

Km (mmol L-1)

Vmax (µmol min-1)

free immobilized: A-Epi A-GA C-Epi C-GA

12.20

4.49

24.13 18.64 30.09 20.76

0.83 1.02 0.97 1.51

enzyme due to chemical boding, diffusion limitation, and confinement of enzyme molecules with polymeric support. For enzyme immobilized by cross-linking techniques, a slight increase in both Km and Vmax demonstrates that the immobilization procedure and the support did not markedly alter the microenvironment of the enzyme molecules probably because of the mild immobilization procedure. References and Notes (1) Bordignon, C.; Notarangelo, L. D.; Servida, P.; Ugazio, A. G.; Mavilio, F. Science 1995, 270, 470. (2) Pool, R. Science 1990, 248, 305. (3) Bradley, C. R.; Rechnitz, G. A. Anal. Chem. 1984, 56, 664. (4) Bradley, C. R.; Rechnitz, G. A. Anal. Chem. 1985, 56, 1401. (5) Silbert, L. S.; Foglia, T. A. Anal. Chem. 1985, 57, 1404. (6) Liu, D.; Meyerhoff, M. E.; Goldberg, H. D.; Brow, R. B. Anal. Chim Acta 1993, 274, 37. (7) Dessouki, A. M.; Issa, G. I.; Atia, K. S. Chem. Technol. Biotechnol. 2001, 76, 700. (8) Lee, K.; Lee, P. M.; Siaw, Y. S. J. Chem. Technol. Biotechnol. 1992, 54, 375. (9) Lowry, O. H.; Rosebrough, N. J.; Farr, J. A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265.

(10) Mohmmed, T. M. Kinetic Investigation of Immobilized Water Melon Citrullus Vulgaris Urease on Cyanuric Chloride Cellulose Ether. Ph.D. Thesis, Cairo University, Cairo, Egypt, 1995. (11) Babu, P.; Venkatram, T. Indian Chem. Eng. 1989, 31, 49. (12) Tomar, M.; Prabhuk, K. Enzymol. Microb. Technol. 1985, 7, 557. (13) Ray, R. R.; Jana, S. C.; Nanda, G. J. Appl. Bacteriol. 1995, 79, 157. (14) Emi, S.; Murase, Y.; Hayashi, T.; Nakajima, A. J. Appl. Polym. Sci. 1990, 41, 2753. (15) Yoshida, M.; Oishi, K.; Kimura, T.; Ogata, M.; Nakakuki, Y. Agric. Biol. Chem. 1989, 35, 3139. (16) Sakhukhan, R.; Roy, S. K.; Chakrabarty, S. L. Enzymol. Microb. Technol. 1987, 9, 550. (17) Sungur, S.; Murat, E.; Akbulut, U. Biomaterials 1992, 13, 795. (18) De Cordt, S.; Saraiva, J.; Hendrikx, M.; Maesmans, G.; Tobback, J. In Stability and Stabilization of Enzymes; Elseiver Science Publishers: London, 1993; p 261. (19) De Cordt, S.; Saraiva, J.; Hendrikx, M.; Maesmans, G.; Tobback. J. Food Sci. Technol. 1992, 27, 661. (20) Kaul, R.; Dsouza, S. F.; Nadkarni, G. Biotechnol. Bioeng. 1984, XXVI, 901. (21) Khare, S. K.; Gupta, M. N. Biotechnol. Bioeng. 1988, 31, 829. (22) Liu, D.; Meyerhoff, M. E. Anal. Chim. Acta 1993, 274, 37. (23) Jacobs, G. P. Radiat. Phys. Chem. 1985, 26, 133. (24) Libicky, A.; Urgan, J.; Chottova, O.; Fidderova, J. Cesk. Farm. 1980, 29, 14. (25) Morimoto, K.; Kimura, S.; Inamori, Y.; Morisaka, K. Chem. Pharm. Bull. 1980, 28, 1304. (26) Abdul-Hamid, I.; Moody, G. J.; Thomas, J. D. Analyst 1988, 113, 81; 1989, 114, 1587; 1990, 115, 1289. (27) Greenfield, P. E.; Laurence, R. L. J. Food Sci. 1975, 40, 906. (28) Ryl;tsev, V. L.; Samoilova, T. I.; Volkouinskaya, L. P.; Bondareva, L. N.; Zazhireri, V. D.; Netrebenko, S. V.; Khachiyant, V. I. Prikl. Biokhim. Mikrobiol. 1984, 20, 694.

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