Back
 AJAC  Vol.4 No.9 , September 2013
Enzyme Substrates Protective Encapsulation within Polymeric Microspheres
Abstract: Determination of enzymes activity is an important task of analytical and biomedical oriented fluorimetry. Despite of a long track record of application, there is still some room for improvements in this field. In the case of hydrolases, spontaneous decomposition of substrate leads to substantial errors in determination of enzyme activity. An innovative and effective approach is proposed allowing protection of enzyme substrate within the lipophilic moiety of polyacrylate microspheres. It is shown that the introduction of substrate into microspheres is not only an effective method of prevention of unwanted spontaneous process, but also does not disturb the availability of substrate for enzymatic hydrolysis. The effect of presence of proteins in the sample on fluorimetric responses was studied. In contrary to previous studies related to application of lipophilic polymers in biomedical analysis, it is shown that the presence of bovine serum albumins enhances the sensitivity of fluorimetric determination. It is shown that this surprising effect is related to adsorption of proteins on the microspheres surface and change of surface charge of polymer.
Cite this paper: J. Zajda, A. Jastrzębska, A. Olszyna, E. Malinowska and A. Michalska, "Enzyme Substrates Protective Encapsulation within Polymeric Microspheres," American Journal of Analytical Chemistry, Vol. 4 No. 9, 2013, pp. 432-441. doi: 10.4236/ajac.2013.49054.
References

[1]   M. Roberts, “Hydrolysis of 4-Methylumbelliferyl Butyrate: A Convenient and Sensitive Fluorescent Assay for Lipase Activity,” Lipids, Vol. 20, No. 4, 1985, pp. 243-247. doi:10.1007/BF02534195

[2]   R. S. Sparkes, S. Targum, E. Gershon, G. F. Sensabaugh, M. C. Sparkes and M. Crist, “Evidence for a Null Allele at the Esterase D (EC 3.1.1.1) Locus,” Human Genetics, Vol. 46, No. 3, 1979, pp. 319-323. doi:10.1007/BF00273315

[3]   E. Nyfeler, J. Grognux, D. Wahler and J.-L. Reymond, “A Sensitive and Selective High-Throughput Screening Fluorescence Assay for Lipases and Esterases,” Helvetica Chimica Acta, Vol. 86, No. 8, 2003, pp. 2919-2927. doi:10.1002/hlca.200390240

[4]   S. Peper and E. Bakker, “Fluorescent Ion-Sensing Microspheres for Multiplexed Chemical Analysis of Clinical and Biological Samples,” Sensor Update, Vol. 13, No. 1, 2004, pp. 83-104. doi:10.1002/seup.200390014

[5]   D. Westover, W. R. Seitz and B. K. Lavine, “Synthesis and Evaluation of Nitrated Poly(4-hydroxystyrene) Microspheres for pH Sensing,” Microchemical Journal, Vol. 74, No. 2, 2003, pp.121-129. doi:10.1016/S0026-265X(02)00178-9

[6]   K. Wygladacz and E. Bakker, “Imaging Fiber Microarray Fluorescent Ion Sensors Based on Bulk Optode Microspheres,” Analytica Chimica Acta, Vol. 532, No. 1, 2005, pp. 61-69. doi:10.1016/j.aca.2004.10.071

[7]   V. Bychkova and A. Shvarev, “Fabrication of Micrometer and Submicrometer-Sized Ion-Selective Optodes vis a Solvent Displacement Process,” Analytical Chemistry, Vol. 81, No. 6, 2009, pp. 2325-2331. doi:10.1021/ac8024619

[8]   M. Bradley, L. Alexander, K. Duncan, M. Chennaoui, A. C. Jones and R. M. Sánchez-Martín, “pH Sensing in Living Cells Using Fluorescent Microspheres,” Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 1, 2008, pp. 313-317. doi:10.1016/j.bmcl.2007.10.075

[9]   H. A. Clark, R. Kopelman, R. Tjalkens and M. A. Philbert, “Optical Nanosensors for Chemical Analysis inside Single Living Cells Part 2: Sensors for pH and Calcium and the Intracellular Application of PEBBLE,” Analytical Chemistry, Vol. 71, No. 21, 1999, pp. 4837-4843. doi:10.1021/ac990630n

[10]   H. A. Clark, M. Hoyer, M. A. Philbert and R. Kopelman, “Optical Nanosensors for Chemical Analysis inside Single Living Cells Part 1: Fabrication, Characterization and Methods for Intracellular Delivery,” Analytical Chemistry, Vol. 71, No. 21, 1999, pp. 4831-4836. doi:10.1021/ac990629o

[11]   E. J. Park, M. Brausel, C. Behrand, M. A. Philbert and R. Kopelman, “Ratiometric Optical PEBBLE Nanosensors for Magnesium Ions in Viable Cells,” Analytical Chemistry, Vol. 75, No. 15, 2003, pp.3784-3791. doi:10.1021/ac0342323

[12]   W. Tang, H. Xu, E. J. Park, M. A. Philbert and R. Kopelman, “Encapsulation of Methylene Blue in Polyacrylamide Nanoparticle Platforms Protects Its Photodynamic Effectiveness,” Biochemical and Biophysical Research Communications, Vol. 369, No. 2, 2008, pp. 579-583. doi:10.1016/j.bbrc.2008.02.066

[13]   J. P. Sumner, J. W. Aylott, E. Monson and R. Kopelman, “A Fluorescent PEBBLE Nanosensor for Intracellular Free Zinc.” Analyst, Vol. 127, No. 1, 2002, pp. 11-16. doi:10.1039/b108568a

[14]   K. L. Brogan and D. R. Walt, “Optical Fiber-Based Sensors: Application to Chemical Biology,” Current Opinion in Chemical Biology, Vol. 9, No. 5, 2005, pp. 494-500. doi:10.1016/j.cbpa.2005.08.009

[15]   A. Michalska and E. A. H. Hall, “Fluorescent Polymeric Spheres for Alkaline Phosphatase Sensing,” Presentation at Pittcon 2009 Conference, 2009.

[16]   M. J. Ruedas-Rama and E. A. H. Hall, “K+-Selective Nanospheres: Maximising Response Range and Minimising Response Time,” Analyst, Vol. 131, No. 12, 2006, pp. 1282-1291. doi:10.1039/b608901a

[17]   L. Y. Heng, K. Toth and E. A. H. Hall, “Ion-Transport and Diffusion Coefficients of Non-Plasticized Methacrylic-Acrylic Ion-Selective Membranes,” Talanta, Vol. 63, No. 1, 2004, pp. 73-87. doi:10.1016/j.talanta.2003.12.051

[18]   A. Michalska, M. Wojciechowski, E. Bulska and K. Maksymiuk, “Quantifying Primary Silver Ions Contents in Poly(vinyl chloride) and Poly(n-butyl acrylate) Ion-Selective Membranes.” Electroanalysis, Vol. 21, No. 17-18, 2009, pp. 1931-1938. doi:10.1002/elan.200804611

[19]   B. Figureurska and J. Pluta, “Badanie Dostepnosci Farmaceutycznej Kreonu® Neo—Pancreatinum i Panzytratu,” Advances in Clinical and Experimental Medicine, Vol. 13, No. 5, 2004, pp. 779-787.

[20]   T. de Laborde de Monpezat, B. de Jeso, J.-L. Butour, L. Chavant and M. Sancholle, “A fluorimetric Method for Measuring Lipase Activity,” Lipids, Vol. 25, No. 10, 1990, pp. 661-664. doi:10.1007/BF02536018

[21]   D. Martin, J. Ruiz, M. Flores and F. Toldra, “Effect of Dietary Conjugated Linoleic Acid and Monounsaturated Fatty Acid Content on Pig Muscle and Adipose Tissue Lipase and Esterase Activity,” Journal of Agricultural and Food Chemistry, Vol. 54, No. 24, 2006, pp. 9241-9247. doi:10.1021/jf061765e

[22]   B. Borgstrom and C. Erlanson, “Interactions of Serum Albumin and Other Proteins with Porcine Pancreatic Lipase,” Gastroenterology, Vol. 75, No. 3, 1978, pp. 382-386.

[23]   Y. Gargouri, G. Pieroni, C. Riviere, A. Sugihara, L. Sandra and R. Verger, “Inhibition of Lipases by Proteins, a Kinetic Study with Dicaprin Monolayers,” The Journal of Biological Chemistry, Vol. 260, No. 4, 1985, pp. 2268-2273.

[24]   H. Chahinian, S. Bezzine, F. Ferrato, M. G. Ivanova, B. Perez, E. M. Lowe and F. Carriere “The β5 Loop of the Pancreatic Lipase C2-Like Domain Plays a Critical Role in the Lipase—Lipid Interactions,” Biochemistry, Vol. 41, No. 46, 2002, pp. 13725-13735. doi:10.1021/bi0257944

[25]   J. C. Khoo, E. J. Vance, E. M. Mahoney, D. Jensen, E. Wancewicz and D. Steinberg, “Neutral Triglyceride Lipase in Macrophages,” Artreriosclerosis, Vol. 4, No. 1, 1984, pp. 34-40. doi:10.1161/01.ATV.4.1.34

[26]   Z. Sroka, “The Activity of Lipase from Rhizopus sp. in Native Form and after Immobilization on Hollow-Fiber Membranes,” Journal of Membrane Science, Vol. 97, 1994, pp. 209-214. doi:10.1016/0376-7388(94)00163-S

[27]   N. W. Tietz and D. F. Shuey, “Lipase in Serum-the Elusive Enzyme: An Overview,” Clinical Chemistry, Vol. 39, No. 5, 1993, pp. 746-756.

[28]   S. S. Kanwar, I. A. Ghazi, S. S. Chimni, G. K. Joshi, G. V. Rao, R. K. Kaushal, R. Gupta and V. Punji, “Purification and Properties of a Novel Extra-Cellular Thermotolerant Metallolipase of Bacillus Coagulants MTCC-6375 Isolate,” Protein Expression and Purification, Vol. 46, No. 2, 2006, pp. 421-428. doi:10.1016/j.pep.2005.10.007

[29]   K. R. Kim, D. Y. Kwon, S. H. Yoon and W. Y. Kim, “Purification, Refolding, and Characterization of Recombinant Pseudomonas Fluorescens Lipase,” Protein Expression and Purification, Vol. 39, No. 1, 2005, pp. 124-129. doi:10.1016/j.pep.2004.09.014

[30]   S. Sabri, R. N. Z. R. A. Rahman, T. L. Leow, M. Basri and A. B. Salleh, “Secretory Expression and Characterization of Highly Ca2+-Activated Thermostable L2 Lipase,” Protein Expression and Purification, Vol. 68, No. 2, 2009, pp. 161-166. doi:10.1016/j.pep.2009.08.002

[31]   J. D. A. Tyndall, S. Sinchaikul, L. A. Forthergill-Gilmore, P. Taylor and M. D. Walkinshaw, “Crystal Structure of a Thermostable Lipase from Bacillus Stearothermophilus P1,” Journal of Molecular Biology, Vol. 323, No. 5, 2002, pp. 859-869. doi:10.1016/S0022-2836(02)01004-5

[32]   U. Bohme and U. Scheler, “Effective Charge of Bovine Serum Albumin Determined by Electrophoresis NMR,” Chemical Physics Letters, Vol. 435, No. 4-5, 2007, pp. 342-345. doi:10.1016/j.cplett.2006.12.068

[33]   J. Donner, “Preparation of Porcine Pancreatic Lipase Free of Co-lipase Activity,” Acta Chemica Scandinavica— Series B, Vol. 30, No. 5, 1976, pp. 430-434. doi:10.3891/acta.chem.scand.30b-0430

 
 
Top