Units and Methods of Proteolytic Activity Determination

Author(s): Rafał Ireneusz Rawski, Przemysław Tomasz Sanecki*, Jan Kalembkiewicz

Journal Name: Current Pharmaceutical Analysis

Volume 16 , Issue 6 , 2020

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Graphical Abstract:


Background: In order to organize and give a better understanding of the existing population of protease activity units together with their respective methods of enzymatic activity assessment, there is a need of their clear classification system.

Results and Conclusion: The following system has been proposed: Enzyme Centered Units (ECU) equivalent to Enzyme Process Unit notation; Protein Centered Units (PCU) equivalent to Protein Process Unit notation; Legal Authority and Enzyme Centered Units (LAECU) equivalent to Enzyme Centered Units system additionally related to a legal authority or an organization. The suitable ways for the mutual conversion of commonly used units and their conversion into the standard SI units have been included. A convenient gravity/spectrophotometer test of proteolytic activity with the use of three protein types has also been proposed. The test gives high degree of confidence of the experimental determination for a wide spectrum of protease activity in samples of plant origin. The whole paper allows both theoretical and practical orientation in the range of different proteolytic activity units as well as in the methods of their determination.

Keywords: Protease, classification, unit, conversion, activity, review.

Lauwers, A.; Scharpé, S.; Swarbrick, J.Series., Eds.; Pharmaceutical Enzymes, 1st ed; Drugs and the pharmaceutical sciences; Marcel Dekker. Inc.: New York, 1997, p. 84.
Mótyán, J.A.; Tóth, F.; Tőzsér, J. Research applications of proteolytic enzymes in molecular biology. Biomolecules, 2013, 3(4), 923-942.
[http://dx.doi.org/10.3390/biom3040923] [PMID: 24970197]
Eriksen, D.T.; Zhao, H.; Tang, L.W.; Nair, N.U. Industrial Applications of Enzymes as Catalysts.Manual of Industrial Microbiology and Biotechnology, 3rd ed; Lynd, L.R.; Zhao, H.; Katz, L.; Baltz, R.H.; Bull, A.T.; Junker, B.; Masurekar, P.; Davies, J.E.; Reeves, C.D.; Demain, A.L., Eds.; ASM Press: Washington, DC, 2010, pp. 480-494.
Vitolo, M. Industrial Uses of Enzymes. In: Fundamentals in Biotechnology; Doelle, H. W.; Rokem, J. S.; Berovic, M., Eds.; BIOTECHNOLOGY. EOLSS Publications: Oxford, England, 2011; 6, pp. 135-219.
Balakireva, A.V.; Kuznetsova, N.V.; Petushkova, A.I.; Savvateeva, L.; Zamyatnin, A.A. Trends and prospects of plant proteases in therapeutics. Curr. Med. Chem., 2017, 25
[http://dx.doi.org/10.2174/0929867325666171123204403] [PMID: 29173148]
Schaller, A. A cut above the rest: the regulatory function of plant proteases. Planta, 2004, 220(2), 183-197.
[http://dx.doi.org/10.1007/s00425-004-1407-2] [PMID: 15517349]
Wegstein, J.; Heinsohn, H. CHAPTER 4 - Modern Methods of Enzyme Expression and Design. In: Enzymes in Food Processing; Nagodawithana, T.; Reed, G., Eds.; Food Science and Technology. Academic Press: London, 1993; pp. 71-101.
Headon, D.R.; Walsh, G. The industrial production of enzymes. Biotechnol. Adv., 1994, 12(4), 635-646.
[http://dx.doi.org/10.1016/0734-9750(94)90004-3] [PMID: 14545919]
Kotlar, C.; Ponce, A.; Roura, S. Characterization of a novel protease from Bacillus cereus and evaluation of an eco-friendly hydrolysis of a brewery byproduct: characterization of a novel protease from Bacillus cereus. J. Inst. Brew., 2015, 121(4), 558-565.
Matsushita, M.; Thiel, S.; Jensenius, J.C.; Terai, I.; Fujita, T. Proteolytic activities of two types of mannose-binding lectin-associated serine protease. J. Immunol., 2000, 165(5), 2637-2642.
[http://dx.doi.org/10.4049/jimmunol.165.5.2637] [PMID: 10946292]
Langdon, T.K. Pepsin as a Case Study for Method and Unit Harmonization; The Spalding Auditorium; USP Headquarters: Rockville, Maryland, 2009.
Wolfson, D.; Olmstead, S.; Meiss, D.; Ralston, J. Making Sense of Digestive Enzymes; Klaire Labs Technical Summary, 2010, pp. 1-8.
Rockwell. Enzymes Standards of Measurement. Rockwell Nutrition Blog, 2011.
ProThera, Inc.. Klaire Labs. DPP-IV Enzymes: Clearing Up the Confusion about Activity Units; Integrated Nutraceuticals for Healthcare Professionals, 2010, pp. 1-2.
Enzymelady. Comparing Digestive Enzyme Products, Enzyme Essentials. 2014.
King, E.J.; Campbell, D.M. International enzyme units. An attempt at international agreement. Clin. Chim. Acta, 1961, 6(3), 301-306.
[http://dx.doi.org/10.1016/0009-8981(61)90054-7] [PMID: 13756069]
Porterfield, A. Catalyzing Through Confusion: Making (Some) Sense of En-zyme Units Available from:. https://bitesizebio.com/24702/catalyzing-through-confusion-making-some-sense-of-enzyme-units/
Nomenclature Committee of the International Union of Biochemistry (NC-IUB). Units of Enzyme Activity. Eur. J. Biochem., 1979, 97(2), 319-320.
Harrison, D. Labeling of Enzyme Dietary Supplements. National Enzyme Company, 2012.
Tipton, K.F.; Armstrong, R.N.; Bakker, B.M.; Bairoch, A.; Cornish-Bowden, A.; Halling, P.J.; Hofmeyr, J-H.; Leyh, T.S.; Kettner, C.; Raushel, F.M. Standards for Reporting Enzyme Data: The STRENDA Consortium: What It Aims to Do and Why It Should Be Helpful. Perspect Sci (Neth), 2014, 1(1–6), 131-137.
Cichoke, A.J. The Complete Book of Enzyme Therapy; Avery Publishing: Garden City Park, N.Y, 1999.
Buttery, J.E. Angiotensin-converting enzyme: confusion about activity units. Clin. Chem., 1985, 31(6), 1071-1072.
[http://dx.doi.org/10.1093/clinchem/31.6.1071] [PMID: 2986879]
King, E.J.; Moss, D.W. International enzyme units and isoenzyme nomenclature. J. Clin. Pathol., 1963, 16(4), 391-393.
[http://dx.doi.org/10.1136/jcp.16.4.391] [PMID: 16811013]
Baltierra-Trejo, E.; Márquez-Benavides, L.; Sánchez-Yáñez, J.M. Inconsistencies and ambiguities in calculating enzyme activity: The case of laccase. J. Microbiol. Methods, 2015, 119, 126-131.
[http://dx.doi.org/10.1016/j.mimet.2015.10.007] [PMID: 26459230]
Stryer, L. Biochemia, 2nd, popr; Augustyniak, J; Michejda, J., Ed.; PWN: Warszawa, 2003.
Martínez Cuesta, S.; Rahman, S.A.; Furnham, N.; Thornton, J.M. The classification and evolution of enzyme function. Biophys. J., 2015, 109(6), 1082-1086.
[http://dx.doi.org/10.1016/j.bpj.2015.04.020] [PMID: 25986631]
Arshad, Z.I.M.; Amid, A.; Yusof, F.; Jaswir, I.; Ahmad, K.; Loke, S.P. Bromelain: an overview of industrial application and purification strategies. Appl. Microbiol. Biotechnol., 2014, 98(17), 7283-7297.
[http://dx.doi.org/10.1007/s00253-014-5889-y] [PMID: 24965557]
Burke, D.E.; Lewis, S.D.; Shafer, J.A. A two-step procedure for purification of papain from extract of papaya latex. Arch. Biochem. Biophys., 1974, 164(1), 30-36.
[http://dx.doi.org/10.1016/0003-9861(74)90004-6] [PMID: 4372948]
Kilara, A.; Shahani, K.M.; Wagner, F.W. Preparation and properties of immobilized papain and lipase. Biotechnol. Bioeng., 1977, 19(11), 1703-1714.
[http://dx.doi.org/10.1002/bit.260191109] [PMID: 21714]
Baeyens-Volant, D.; Matagne, A.; El Mahyaoui, R.; Wattiez, R.; Azarkan, M. A novel form of ficin from Ficus carica latex: Purification and characterization. Phytochemistry, 2015, 117, 154-167.
[http://dx.doi.org/10.1016/j.phytochem.2015.05.019] [PMID: 26083455]
Devaraj, K.B.; Kumar, P.R.; Prakash, V. Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica. J. Agric. Food Chem., 2008, 56(23), 11417-11423.
[http://dx.doi.org/10.1021/jf802205a] [PMID: 18991449]
Boland, M. Chapter Four - Kiwifruit Proteins and Enzymes: Actinidin and Other Significant Proteins. In: Nutritional Benefits of Kiwifruit; Moughan, P. J., Ed.; Advances in Food and Nutrition Research. Academic Press: Cambridge, 2013; 68, pp. 59-80.
Rutherfurd, S.M.; Montoya, C.A.; Zou, M.L.; Moughan, P.J.; Drummond, L.N.; Boland, M.J. Effect of Actinidin from Kiwifruit (Actinidia deliciosa Cv. Hayward) on the digestion of food proteins determined in the growing rat. Food Chem., 2011, 129(4), 1681-1689.
Beilinson, V.; Moskalenko, O.V.; Livingstone, D.S.; Reverdatto, S.V.; Jung, R.; Nielsen, N.C. Two subtilisin-like proteases from soybean. Physiol. Plant., 2002, 115(4), 585-597.
[http://dx.doi.org/10.1034/j.1399-3054.2002.1150413.x] [PMID: 12121465]
Arima, K.; Uchikoba, T.; Yonezawa, H.; Shimada, M.; Kaneda, M. Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai. Phytochemistry, 2000, 54(6), 559-565.
[http://dx.doi.org/10.1016/S0031-9422(00)00075-3] [PMID: 10963447]
Nishikata, M. Trypsin-like protease from soybean seeds. Purification and some properties. J. Biochem., 1984, 95(4), 1169-1177.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a134706] [PMID: 6378896]
Rawski, R.I.; Sanecki, P.T.; Dżugan, M.; Kijowska, K. The evidence of proteases in sprouted seeds and their application for animal protein digestion. Chem. Zvesti, 2018, 72(5), 1213-1221.
[http://dx.doi.org/10.1007/s11696-017-0341-2] [PMID: 29681684]
Fujita, M.; Nomura, K.; Hong, K.; Ito, Y.; Asada, A.; Nishimuro, S. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan. Biochem. Biophys. Res. Commun., 1993, 197(3), 1340-1347.
[http://dx.doi.org/10.1006/bbrc.1993.2624] [PMID: 8280151]
Wang, S-L.; Wu, Y-Y.; Liang, T-W. Purification and biochemical characterization of a nattokinase by conversion of shrimp shell with Bacillus subtilis TKU007. N. Biotechnol., 2011, 28(2), 196-202.
[http://dx.doi.org/10.1016/j.nbt.2010.09.003] [PMID: 20849993]
Miyata, K.; Maejima, K.; Tomoda, K.; Isono, M. serratia protease: part i. purification and general properties of the enzyme. Biosci. Biotechnol. Biochem., 1970, 34(2), 310-318.
Seijffers, M.J.; Miller, L.L.; Segal, H.L. Partial characterization of human pepsin i, pepsin iia, pepsin iib, and pepsin 3. Biochemistry, 1964, 3, 1203-1209.
[http://dx.doi.org/10.1021/bi00897a003] [PMID: 14229659]
Pepsin, A. BRENDA (The Comprehensive Enzyme Information System); Technische Universität Braunschweig, 2018,.
McDowall, J. Trypsin and Chymotrypsin, Available from:. https://ebi.ac.uk/interpro/potm/2003_5/Page1.htm
Kolsrud, H.; Malerod, H.; Ray, S.; Reubsaet, L.; Lundanes, E.; Greibrokk, T. A Critical Review of Trypsin Digestion for LC-MS Based Proteomics.Integrative Proteomics. Leung, H-C. In-Tech: Rijeka, 2012, pp. 4-92.
Worthington Biochemical Corporation. Manual of Clinical Enzyme Measurements; Worthington Diagnostics: Lakewood, NJ, 1972.
[47] Chymotrypsin. BRENDA (The Comprehensive Enzyme Information System); Technische Universität Braunschweig, 2018.
Mihara, H.; Sumi, H.; Yoneta, T.; Mizumoto, H.; Ikeda, R.; Seiki, M.; Maruyama, M. A novel fibrinolytic enzyme extracted from the earthworm, Lumbricus rubellus. Jpn. J. Physiol., 1991, 41(3), 461-472.
[http://dx.doi.org/10.2170/jjphysiol.41.461] [PMID: 1960890]
Cho, I.H.; Choi, E.S.; Lim, H.G.; Lee, H.H. Purification and characterization of six fibrinolytic serine-proteases from earthworm Lumbricus rubellus. J. Biochem. Mol. Biol., 2004, 37(2), 199-205.
[PMID: 15469696]
Scocca, J.; Lee, Y.C. The composition and structure of the carbohydrate of pineapple stem bromelain. J. Biol. Chem., 1969, 244(18), 4852-4863.
[PMID: 4309986]
Dybkær, R. Unit “Katal” for Catalytic Activity (IUPAC Technical Report). Pure Appl. Chem., 2001, 73(6), 927-931.
The Joint Commission Facts about the Official “Do Not Use” List of Abbreviations., 2017. June 9
Lager, R.K. A new method for the assay of papain. Ann. N. Y. Acad. Sci., 1951, 54(2), 236-244.
[http://dx.doi.org/10.1111/j.1749-6632.1951.tb39917.x] [PMID: 14830114]
Worthington Biochemical Corporation. Papain Assay, Available from:. http://worthington-biochem.com/pap/assay.html
Khan, M.A.; Ahmad, N.; Zafar, A.U.; Nasir, I.A.; Qadir, M.A. Isolation and screening of alkaline protease producing bacteria and physio-chemical characterization of the enzyme. AJB, 2011, 10(33), 10.
Horikoshi, K. Production of alkaline enzymes by alkalophilic microorganisms: part i. alkaline protease produced by Bacillus no. 221 Biosci. Biotechnol. Biochem., 1971, 35(9), 1407-1414.
Boirie, Y.; Dangin, M.; Gachon, P.; Vasson, M-P.; Maubois, J-L.; Beaufrère, B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc. Natl. Acad. Sci. USA, 1997, 94(26), 14930-14935.
[http://dx.doi.org/10.1073/pnas.94.26.14930] [PMID: 9405716]
Dangin, M.; Boirie, Y.; Garcia-Rodenas, C.; Gachon, P.; Fauquant, J.; Callier, P.; Ballèvre, O.; Beaufrère, B. The digestion rate of protein is an independent regulating factor of postprandial protein retention. Am. J. Physiol. Endocrinol. Metab., 2001, 280(2), E340-E348.
[http://dx.doi.org/10.1152/ajpendo.2001.280.2.E340] [PMID: 11158939]
Rick, W. Methoden der enzymatischen Analyse, 3rd ed; Bergmeyer, H.U., Ed.; Verlag Chemie: Weinheim, FRG, 1974.
Cupp-Enyard, C. Sigma’s Non-specific Protease Activity Assay - Casein as a Substrate. J. Vis. Exp., 2008, (19), 899.
[http://dx.doi.org/10.3791/899] [PMID: 19066538]
Reimerdes, E.H.; Klostermeyer, H. Determination of Proteolytic Activities on Casein Substrates. In: Proteolytic enzymes. Pt. B: Proteolytic enzymes; Lorand, L.; Perlmann, G. E., Eds.; Methods in enzymology. Academic Press: New York, NY, 1976; pp. 26-27.
Rodarte, M.P.; Dias, D.R.; Vilela, D.M.; Schwan, R.F. Proteolytic activities of bacteria, yeasts and filamentous fungi isolated from Coffee Fruit (Coffea arabica L.). Acta Sci. Agron., 2011, 33(3)
Christensen, L.R. The action of proteolytic enzymes on casein proteins. Arch. Biochem. Biophys., 1954, 53(1), 128-137.
[http://dx.doi.org/10.1016/0003-9861(54)90240-4] [PMID: 13208290]
Yamauchi, K.; Kaminogawa, S. Decomposition of milk proteins by milk protease. Agric. Biol. Chem., 1972, 36(2), 249-254.
Twining, S.S. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Anal. Biochem., 1984, 143(1), 30-34.
[http://dx.doi.org/10.1016/0003-2697(84)90553-0] [PMID: 6442109]
Charney, J.; Tomarelli, R.M. A colorimetric method for the determination of the proteolytic activity of duodenal juice. J. Biol. Chem., 1947, 171(2), 501-505.
[PMID: 20272088]
Coêlho, D.F.; Saturnino, T.P.; Fernandes, F.F.; Mazzola, P.G.; Silveira, E.; Tambourgi, E.B. Azocasein substrate for determination of proteolytic activity: reexamining a traditional method using bromelain samples. BioMed Res. Int., 2016, 2016, 8409183
[http://dx.doi.org/10.1155/2016/8409183] [PMID: 26925415]
Iversen, S.L.; Jørgensen, M.H. azocasein assay for alkaline protease in complex fermentation broth. Biotechnol. Tech., 1995, 9(8), 573-576.
Boonacker, E.; Van Noorden, C.J.F. Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis. J. Histochem. Cytochem., 2001, 49(12), 1473-1486.
[http://dx.doi.org/10.1177/002215540104901201] [PMID: 11724895]
Jones, L.J.; Upson, R.H.; Haugland, R.P.; Panchuk-Voloshina, N.; Zhou, M.; Haugland, R.P. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal. Biochem., 1997, 251(2), 144-152.
[http://dx.doi.org/10.1006/abio.1997.2259] [PMID: 9299009]
Schickaneder, E.; Hösel, W.; Eltz, H.d.; Geuß, U. casein-resorufon, a new substrate for a highly sensitive protease assay. Fresenius J. Anal. Chem., 1988, 330(4–5), 360-360.
Jinka, R.; Ramakrishna, V.; Rao, S.K.; Rao, R.P. Purification and characterization of cysteine protease from germinating cotyledons of horse gram. BMC Biochem., 2009, 10(1), 28-39.
[http://dx.doi.org/10.1186/1471-2091-10-28] [PMID: 19919695]
Sarath, G. Dela motte, R. S.; Wagner, F. W Proteolytic Enzyme. A Practical Approach; IRL Press: Oxford, England, 1989.
Gray, V.A.; Cole, E.; Riva Toma, J.M.D.; Ghidorsi, L.; Guo, J-H.; Han, J-H.; Han, F.; Hosty, C.T.; Kochling, J.D.; Kraemer, J. Use of enzymes in the dissolution. testing of gelatin capsules and gelatin-coated tablets-revisions to dissolution and disintegration and dissolution of dietary supplements. Dissolut. Technol., 2014, 21(4), 6-18.
Enzyme Development Corporation. Gelain Digestion Unit Analytical Method (GDU) Available from:. http://enzymedevelopment.com/wp-content/uploads/2011/10/Gelatin-Digestion-Unit-GDU.pdf
Kotb, E. Activity assessment of microbial fibrinolytic enzymes. Appl. Microbiol. Biotechnol., 2013, 97(15), 6647-6665.
[http://dx.doi.org/10.1007/s00253-013-5052-1] [PMID: 23812278]
Astrup, T.; Müllertz, S. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys., 1952, 40(2), 346-351.
[http://dx.doi.org/10.1016/0003-9861(52)90121-5] [PMID: 12997222]
Sumi, H.; Hamada, H.; Tsushima, H.; Mihara, H.; Muraki, H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia, 1987, 43(10), 1110-1111.
[http://dx.doi.org/10.1007/BF01956052] [PMID: 3478223]
Sumi, H. Manual of Functional Food Material, 1990, 287-289.
Cheng, G.; He, L.; Sun, Z.; Cui, Z.; Du, Y.; Kong, Y. Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3. J. Microbiol. Biotechnol., 2015, 25(9), 1449-1459.
[http://dx.doi.org/10.4014/jmb.1503.03015] [PMID: 26017226]
Gidron, E.; Margalit, R.; Shalitin, Y. A rapid screening test for reduced fibrinolytic activity of plasma: streptokinase activated lysis time. J. Clin. Pathol., 1978, 31(1), 54-57.
[http://dx.doi.org/10.1136/jcp.31.1.54] [PMID: 627618]
Chakrabarti, R.; Fearnley, G.R. The ‘fibrinolytic potential’ as a simple measure of spontaneous fibrinolysis. J. Clin. Pathol., 1962, 15, 228-230.
[http://dx.doi.org/10.1136/jcp.15.3.228] [PMID: 13877892]
Thomson, J.M., Ed.; Blood Coagulation and Haemostasis. A Practical Guide, 4th ed; Churchill Livingstone: Edinburgh, 1992, Vol. 3, .
Blix, S. The effectiveness of activators in clot lysis, with special reference to fibrinolytic therapy. A new method for determination of preformed clot lysis. Acta Med. Scand. Suppl., 1962, 386(S386), 1-24.
[http://dx.doi.org/10.1111/j.0954-6820.1962.tb03046.x] [PMID: 13971658]
Howell, M. A method for assessing clot lysis. J. Clin. Pathol., 1964, 17, 310-312.
[http://dx.doi.org/10.1136/jcp.17.3.310] [PMID: 14159468]
Sherry, S.; Alkjaersig, N.; Fletcher, A. P. Activity of plasmin and streptokinase-activator on substituted arginine and lysine esters. Thromb. Haemost, 1966, 16(02), 018-031.
Kessner, A.; Troll, W. Fluorometric microassay of plasminogen activators. Arch. Biochem. Biophys., 1976, 176(2), 411-416.
[http://dx.doi.org/10.1016/0003-9861(76)90183-1] [PMID: 185962]
Niinobe, M.; Hitomi, Y.; Fujii, S. A sensitive colorimetric assay for various proteases using naphthyl ester derivatives as substrates. J. Biochem., 1980, 87(3), 779-783.
[http://dx.doi.org/10.1093/oxfordjournals.jbchem.a132807] [PMID: 6446549]
Sweetman, S.C., Ed.; Martindale: The Complete Drug Reference, 37th ed; Pharmaceutical Press: London, England, UK, 2011.
Kuhn, R.J.; Eyting, S.; Henniges, F.; Potthoff, A. In vitro comparison of physical parameters, enzyme activity, acid resistance, and pH dissolution characteristics of enteric-coated pancreatic enzyme preparations: implications for clinical variability and pharmacy substitution. J. Pediatr. Pharmacol. Ther., 2007, 12(2), 115-128.
[http://dx.doi.org/10.5863/1551-6776-12.2.115] [PMID: 23055848]
Lauwers, A.; Uyttenbroeck, W.; Samyn, N. Pancreatic Enzyme Replacement. In: Pharmaceutical enzymes; Lauwers, A.; Scharpé, S.; Swarbrick, J., Series., Eds.; Drugs and the pharmaceutical sciences. Marcel Dekker, Inc.: New York, 1997; 84, pp. 187-221.
Neidert, L.E.; Wainright, K.S.; Zheng, C.; Babu, J.R.; Kluess, H.A. Plasma dipeptidyl peptidase IV activity and measures of body composition in apparently healthy people. Heliyon, 2016, 2(4) e00097
[http://dx.doi.org/10.1016/j.heliyon.2016.e00097] [PMID: 27441271]
Wikström, M.; Elwing, H.; Linde, A. Determination of proteolytic activity: a sensitive and simple assay utilizing substrate adsorbed to a plastic surface and radial diffusion in gel. Anal. Biochem., 1981, 118(2), 240-246.
[http://dx.doi.org/10.1016/0003-2697(81)90185-8] [PMID: 6802022]

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Year: 2020
Published on: 01 July, 2020
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