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US005830741A United States Patent [19] [11] Patent Number: 5,830,741 Dwulet et al. [45] Date of Patent: *Nov. 3, 1998
[54] COMPOSITION FOR TISSUE DISSOCIATION Suggs et al. (1992) J. Vasc. Surg., 15(1), “Enzymatic Har CONTAINING COLLAGENASE I AND II vesting of Adult Human Saphenous Vein Endothelial Cells: FROM CLOSTRIDI UM HISTOLYTI C UM AND A Use Of Chemically De?ned Combination of TWo Puri?ed NEUTRAL PROTEASE Enzymes to Attain Viable Cell Yields Equal to Those Attained by Crude Bacterial Collagenase Preparations”, pp. [75] Inventors: Francis E. DWulet, Greenwood; 205—213. Marilyn E. Smith, McCordsville, both Bond et al., “Characterization of the Individual Collagenases of Ind. from Clostridium histolyticum”, 1984, pp. 3085—3091, Bio chemistry vol. 23, No. 13. [73] Assignee: Boehringer Mannheim Corporation, Bond et al., “Puri?cation and Separation of Individual Indianapolis, Ind. Collagenases of Clostridium histolyticum Using Red Dye Ligand Chromatography”, 1984, pp. 3077—3085, Biochem. [ * ] Notice: The term of this patent shall not extend vol. 23 No. 13. beyond the expiration date of Pat. No. Dean et al., “Protein Puri?cation Using Immobilized Trizine 5,753,485. Dyes”, 1979, pp. 301—319, Journal of Chromatography, 165. [21] Appl. No.: 760,893 Emo d et al., “Five Sepharose—Bound Ligands for the Chromatographic Puri?cation of Clostridium Collagenase [22] Filed: Dec. 6, 1996 and Clostripain”, 1977, pp. 51—56, FEBS Letters vol. 77 No. [51] Int. Cl.6 C12N 9/52; C12N 9/00 1. Hatton et al., “The role of proteolytic enzymes derived from [52] US. Cl...... 435/220; 435/183 [58] Field of Search ...... 435/220, 183 crude bacterial collagenase in the liberation of hepatocytes from rat liver”, 1983, pp. 311—318, J.Biochem. 137. [56] References Cited He?ey et al., “Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase”, 1981, pp. U.S. PATENT DOCUMENTS C234—C238, Am. J. Physiology 240. 3,201,325 8/1965 Barton ...... 435/220 He?ey et al., “Enzymatic Isolation of Cells from Neonatal 3,705,083 12/1972 Chiulli et al...... 435/220 Calvaria Using TWo Puri?ed Enzymes from Clostridium 3,821,364 6/1974 Chiulli et al...... 424/9467 histolyticum”, 1983, pp. 227—236, Experimental Cell 4,256,836 3/1981 IsoWa et al...... 435/68.1 Research 149. 4,431,544 2/1984 Atkinson et al. . 210/635 He?ey, “Utilization of FPLC—Puri?ed Bacterial Collagenase 4,431,546 2/1984 Hughes et al. 210/656 from the Isolation of Cells from Bone”, 1987, pp. 505—516, 4,732,758 3/1988 Hurion et al. .. 424/94.2 Journal of Bone and Mineral Research vol. 2 No. 7. 4,797,213 1/1989 Parisius et al. 210/651 Kobayashi et al., “Puri?cation and Characterization of Myo 4,868,121 9/1989 Scharp et al. 435/268 sin from Bovine Thyroid”, Nov. 25, 1977, pp. 8285—8291, 4,873,359 10/1989 Chmurny et al. . 560/40 4,946,792 8/1990 O’Leary ...... 435/268 The Journal of Biological Chemistry, vol. 252, No. 22. 5,079,160 1/1992 Lacy et al. 435/381 Kono, “Puri?cation and Partial Characterization of Col 5,116,615 5/1992 Gokcen et al. .. 424/94.2 lagenolytic Enzymes from Clostridium histolyticum”, Mar. 5,120,656 6/1992 O’Leary et al. .. 435/1.1 1968, pp. 1106—1114, Biochemistry vol. 7 No. 3.
5,173,295 12/1992 Wehling ...... 424/9467 Kula et al., “Consecutive Use of 00—Aminoa1kylagaroses. 5,177,017 1/1993 Lin et al. .. 435/252.33 Res. and Purif. of Clostripain and Collagenase from C. 5,273,904 12/1993 Langley ...... 435/283.1 Histolyticum ”, 1 976, pp. 38 9—396, 5,322,790 6/1994 Scharp et al. . 435/268 Bioch.&Bioph.Res. Comm. vol. 69 No.2. 5,332,503 7/1994 Lee etal...... 210/635 McShane et al., “Protease Activity in Pancreatic Islet Iso 5,422,261 6/1995 Lee et al...... 435/219 lation by Enzymatic Digestion”, 1989, pp. 126—128, Dia FOREIGN PATENT DOCUMENTS betes vol. 38 Suppl. 1. 0 191 613 8/1986 European Pat. Off. . (List continued on next page.) 91/14447 10/1991 WIPO . 96/00283 1/1996 WIPO . Primary Examiner—Jon P. Weber Attorney, Agent, or Firm—Brent A. Harris; D. Michael OTHER PUBLICATIONS Young; Marilyn L. Amick Tavakol et al. (1984) Surgical Forum, 37, “Enhanced Dis [57] ABSTRACT solution of Nucleus Pulposus: Combined Enzyme Approach”, pp. 491—494. An enzyme composition for dissociating tissues is disclosed. Maruyama et al. (1987) J. Pharmacol. Meth., 18, “Prepara The composition comprises puri?ed collagenase I and col tion of Single Smooth Muscle Cells from Guinea Pig Taenia lagenase II from Clostridium histolyticum in a mass ratio of C011 by Combination of Puri?ed Collagenase and Papain”, about 0.3 to 0.6 and a neutral protease such that the ratio of pp. 151—161. total FITC casein units of activity to Wunsch units of activity Maruyama et al. (1988) J. Pharmacol. Meth., 19, “Improve in the composition is about 250:1 to about 600:1. The ment of a Procedure for Preparing Single Smooth Muscle preferred neutral protease is thermolysin. Cells from Guinea Pig Taenia C011 by Puri?ed Collagenase and Papain”, pp. 155—164. 6 Claims, No Drawings 5,830,741 Page 2
OTHER PUBLICATIONS Wolters et al., “An analysis of the role of collagenase and Patel, “Biotechnology: Applications and Research”, 1985, protease in the enzymatic dissociaiton of rat pancreas for pp. 534—5 62, published by Technornic Publishing Company, islet isolation”, 1992, pp. 735—742, Diabetologia 35. Inc., Lancaster Pennsylvania. Sharefkin et al., “Adult human endothelial cell enZyrnatic Wolters et al., “Different Roles of Class I and Class II harvesting”, 1986, pp. 567—577, Journal of Vascular Sur Clostridium Histolyticum Collagenase in Rat Pancreatic gery vol. 4 No. 6. Islet Isolation”, 1995, pp. 227—233, Diabetes vol. 44. Silink et al., “y—Glutarnyl Hydrolase (Conjugase)”, Aug. 10, 1975, pp. 5982—5994, The Journal ofBiological Chemistry, Worthington, “Worthington EnZyrne Manual—enZyrnes and vol. 250, No. 15. related biochernicals”, 1988, pp. 93—101, Worthington Bio Stevens et al., “Dissociation of Lung Tissue into Monodis persed Cells by Use of Puri?ed Collagenase”, 1979, pp. chernical Corporation. 460—470, J. Appl. Biochem. 1. H.O. Jauregui, et al., “Primary Cultures of Rat Hepatocytes Van Wart et al., “Cornplernentary Substrate Speci?cities of Class I and Class II Collagenases from C lostria'ium histolyti in HolloW Fiber Charnbers”, In Wtro Cell Dev. Biol, 30A, cum ”, 1985, pp. 6520—6526, Biochemistry vol. 24 No. 23. 23—29 (1994). 5,830,741 1 2 COMPOSITION FOR TISSUE DISSOCIATION the presence of de?ned amounts of both C. histolyticum CONTAINING COLLAGENASE I AND II collagenase class I (collagenase I) and collagenase class II FROM CLOSTRIDI UM HISTOLYTIC UM AND A (collagenase II) enZymes With at least tWo neutral proteases NEUTRAL PROTEASE has been found to in?uence the efficacy of pancreatic islet isolation. HoWever, there still exists a need for identifying FIELD OF THE INVENTION optimiZed enZyme compositions Which provide for rapid This invention relates to compositions for the enzymatic dissociation of tissue and recovery of a greater number of dissociation of extracellular tissue matrices to alloW tissue viable cells. remodeling and the ef?cient isolation of viable cells and cell SUMMARY OF THE INVENTION clusters from tissue. 10 The present invention provides an enZyme composition BACKGROUND OF THE INVENTION prepared by combining de?ned masses of puri?ed proteases, The enZymatic dissociation of tissue into individual cells including collagenase I and collagenase II from C. histolyti and cell clusters is useful in a Wide variety of laboratory, cum and an amount of an endoprotease such that the ratio of diagnostic and therapeutic applications. These applications 15 the total FITC casein units of activity of the enZyme com involve the isolation of many types of cells for various uses, position to the Wunsch units of activity of the masses of including microvascular endothelial cells for small diameter collagenase I and collagenase II is about 85:1 to about synthetic vascular graft seeding, hepatocytes for gene 3,900:1. When used in tissue dissociation protocols for cell therapy, drug toxicology screening and extracorporeal liver isolation the resulting enZyme composition functions to assist devices, chondrocytes for cartilage regeneration, and 20 effect improved, more rapid dissociation of extracellular islets of Langerhans for the treatment of insulin-dependent matrices and it alloWs recovery of higher yields of the diabetes mellitus. EnZyme treatment Works to fragment desired cells With improved viability relative to the number extracellular matrix proteins and proteins Which maintain and viability of cells harvested using enZyme compositions cell-to-cell contact. Since collagen is the principle protein of the prior art, including crude collagenase compositions. component of tissue ultrastructure, the enZyme collagenase In another embodiment of this invention there is provided has been frequently used to accomplish the desired tissue an enZyme composition consisting essentially of collagenase disintegration. I and collagenase II from C. histolyticum and an Different forms of crude bacterial collagenase derived endoprotease, Wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase from Clostridium histolyticum are commercially available 30 and are used to dissociate cells and cell clusters from tissue. II in the enZyme composition is about 0.3 to about 0.6. The These crude collagenases, derived from cell culture resulting enZyme composition is also an improvement over supernatants, typically contain a mixture of protease previously knoWn compositions because of its ability to enZymes (exhibiting both collagenolytic and non-speci?c more rapidly dissociate extracellular tissue matrices and alloW recovery of a larger number of viable cells. proteolytic activities) and non-protease components (e.g., 35 fermentation by-products, media components, pigments, This invention also provides a method of preparing an other enZymes such as phospholipase, and endotoxins). enZyme composition adapted for isolating living cells from Analysis of commercially-available crude collagenases tissue Which includes the step of combining knoWn masses has shoWn extreme variations in the concentration and ratios of collagenase I and collagenase II from C. histolyticum and of the protease and non-protease components. Such compo 40 an amount of an endoprotease sufficient to raise the total sitional variability is re?ected as Well in the variability and FITC casein activity of the enZyme composition to a level consequent lot-to-lot unpredictability of collagenase product such that the ratio of the total FITC casein activity of the performance in tissue dissociation protocols. In addition to enZyme composition to the total Wunsch unit activity of the this inherent activity variability, each lot of commercial masses of collagenase I and collagenase II in the enZyme collagenase loses activity and performance characteristics 45 composition is about 85:1 to about 3,900:1. over time. Finally, in addition to these problems associated With compositional variability, the use of crude collagenases DETAILED DESCRIPTION OF THE in cell harvest/tissue dissociation protocols usually results in INVENTION less-than-desired results in terms of recovery of cell This invention is based on the discovery that collagenase viability, cell number and cell function. These problems are I and collagenase II from C. histolyticum may be combined particularly signi?cant Where the cells are targeted for use in With de?ned amounts of an endoprotease to provide a novel transplantation or for monitoring the impact of effector enZyme composition having activity pro?les optimal for molecules on cell function. For example, it has been dem isolating cells from donor organ tissue. More speci?cally, it onstrated that the ef?cacy of islet transplantation is depen has been discovered that optimal enZymatic degradation of dent in part on the mass of islets and their viability. In 55 extracellular tissue matrices and concomitant ef?cient lib addition, the drug detoxi?cation function of recovered hepa eration of viable cells and cell clusters from organ tissue can tocytes is signi?cantly impaired by damage to the cells be achieved by using a collagenase enZyme composition occurring during liver tissue dissociation and cell isolation. prepared by adjusting the components of the composition so For most uses of recovered cells it is critical for optimum that the ratio of the total FITC casein activity of the enZyme cell performance that damage to the recovered cells and cell 60 composition to the Wunsch units of activity of the masses of clusters be minimiZed. collagenase I and collagenase II in the composition is about Skilled practitioners have recogniZed the importance of 85:1 to about 3,900:1 and is preferably about 130:1 to about the consistent/predictable activity of protease enZymes used 1,400:1. In one embodiment of the invention, there is in tissue dissociation protocols for ef?cacious cellular iso provided an enZyme composition useful for efficient disso lation (i.e., maintaining cellular integrity, recovering larger 65 ciation of rat liver tissue to provide a high yield of hepato cell clusters and more cells or cell clusters). Speci?cally, the cytes. That enZyme composition includes amounts of colla purity of collagenase compositions and the desirability of genase I and collagenase II from C. histolyticum and an 5,830,741 3 4 endoprotease such that the ratio of the total FITC casein of Peyronie’s disease and reformation of abnormal or her activity of the enzyme composition to the Wunsch units of niated discs. In general, the composition of the present activity of the masses of collagenase I and collagenase II in invention is useful for any application Where the removal of the composition is about 250:1 to about 600:1, With an cells or modi?cation of an extracellular matrix are desired. average ratio of about 400:1. For some applications, it may be preferred that the Another aspect of the present invention is optimizing enZyme composition be substantially free of endotoxins. It mass ratios of the tWo component collagenase enZymes in may also be desirable for the enZyme composition to be enZyme compositions of this invention consisting essentially substantially free of the enZyme clostripain, although gen of collagenase I, collagenase II and an endoprotease. The erally clostripain does not seem to affect performance of the optimal mass ratio of collagenase II to the total collagenase 10 enZyme composition. These undesirable components, Which in the composition, collagenase II (collagenase are both present in crude collagenase obtained from C. I+collagenase II), is about 0.3 to about 0.6. When collage histolyticum, can be separated from the puri?ed collagenase nase I and collagenase II from C. histolyticum are combined I and II components using one or more of the puri?cation in this mass ratio With an endoprotease, the resulting enZyme methods described above. “Substantially free” as used composition is particularly useful to recover cells and cell 15 herein to describe this invention means that effort is or has clusters from tissue samples in greater number, With higher been made to exclude referenced impurity from the compo viability, and With improved reproducibility over tissue sition and that such impurity, if present at all in the dissociation protocols using previously knoWn collagenase composition, is present in but trace amounts, typically less enZyme compositions. In one enZyme composition embodi than 2% by Weight, more typically less than 1% by Weight ment of this invention useful for dissociating rat liver, the 20 and in any event less than an amount Which Would affect the mass ratio of collagenase II to the total collagenase in the aggregate functionality of the composition. composition is about 0.35 to about 0.45. EnZyme compositions of this invention may be prepared The enZyme compositions of the present invention by mixing either a speci?c number of activity units (i.e., include both collagenase I and collagenase II from C. Wunsch units of collagenase I and collagenase II) or speci?c histolyticum. Collagenase I and collagenase II are puri?ed 25 masses of the preferably puri?ed enZymes. FolloWing are from C. histolyticum crude collagenase by a variety of enZyme assays for collagenase, neutral protease and clos methods knoWn to those skilled in the art, including dye tripain that Were used to de?ne the speci?c activities of the ligand af?nity chromatography, heparin affinity puri?ed collagenase components as Well as the total activity chromatography, ammonium sulfate precipitation, hydroxy (FITC) of the entire enZyme composition. Those skilled in lapatite chromatography, siZe exclusion chromatography, 30 the art Will recogniZe that enZyme assays other than those ion exchange chromatography, and metal chelation chroma disclosed beloW may also be used to de?ne and prepare tography. Crude collagenase is commercially available from functionally equivalent enZyme compositions. many sources (e.g., Collagenase P from Boehringer Man nheim Corporation). Collagenase Activity Assay 35 The endoprotease component of the present enZyme com Collagenase activity Was measured using a synthetic position can be any endoprotease, including a serine pro peptide substrate according to the method of Wunsch & tease (e.g., trypsin, chymotrypsin, elastase), a cysteine pro Heidrich (Z. Physiol Chem. 1963; 333:149). This is a tease (e.g., papain, chymopapain) and is preferably a neutral standard method Well knoWn to those skilled in the art of protease (e.g., C. histolyticum neutral protease, dispase, or 40 collagenase puri?cation. The measured activity of collage thermolysin (all EC 3.4.24.4). (EC stands for EnZyme Com nase I using the Wunsch peptide as a substrate typically mission classi?cation. EC 3.4.24.4 is the classi?cation for ranged from 0.2 to 0.6 units (U)/milligram (mg) and Was microbial metalloproteinases.) more typically 0.3 to 0.5 U/mg. The measured activity of For dissociation of a 6 to 12 gram rat liver to recover collagenase II using the Wunsch peptide as a substrate hepatocytes, the activity of the masses of collagenase I and 45 typically ranged from 9.5 to 13.5 U/mg and Was more collagenase II in the enZyme composition is about 7.7 to typically 11 to 13 U/mg. One Wunsch unit (U) of activity is about 62.7 Wunsch units, preferably about 15.1 to about de?ned by the hydrolysis of 1 micromole (,umol) peptide per 52.6 Wunsch units, and more preferably about 24 to about 33 minute at 25° C., pH 7.1. Adescription of the test procedure Wunsch units With an average activity of about 30 Wunsch used for the collagenase activity assay is given beloW. units. The total FITC casein activity of this enZyme com 50 Sample Dilution Buffer (100 millimoles (mM)/liter (l) position is about 5,500 to about 29,700 FITC casein units, tris-(hydroxymethyl)-aminomethane (Tris), pH 7.1): preferably about 7,000 to about 22,000 FITC casein units, 1.21 gram (g) Tris Was dissolved in deioniZed (DI) and more preferably about 8,500 to about 14,500 FITC Water. The pH Was adjusted to 7.1 With 1.0 Normal (N) casein units With an average activity of about 12,000 FITC hydrochloric acid (HCl) and the volume Was adjusted casein units. Accordingly, the ratio range of FITC casein 55 to 100 milliliters (ml) With DI Water. units of the composition to Wunsch units of the composition PZ-Pro-Leu-Gly-Pro-D-Arg substrate: Each blank or test is 5,500/62.7 to 29,700/7.7 or about 85:1 to about 3,900:1, sample Was assayed in duplicate. Total amount of preferably 7,000/52.6 to 22,000/ 15 .1 or about 130:1 to about substrate needed for assay=2 mg of substrate for each 1,400: 1, and more preferably 8,500/33 to 14,500/24 or about test performed, plus 2 mg of substrate extra. (For 250:1 to about 600:1 With an average activity ratio of 60 example, 2 samples tested in duplicate+duplicate blank 12,000/30 or about 400:1. tubes=6 assay tubes. The target amount of substrate In addition to the dissociation of liver for isolation of needed is 14.0 mg.) The total amount of substrate hepatocytes, the above composition is useful for the disso Weighed out Was divided by 50 to determine the ciation of epidermal fat for the isolation of microvascular number of milliliters of methanol needed to dissolve endothelial cells. The above composition is also expected to 65 the substrate. The calculated amount of methanol Was be useful for dissociating pancreas tissue, topical treatment added to the substrate. The substrate Was mixed thor for burn and ulcer clearing and for Wound healing, treatment oughly by vortexing until totally dissolved. Sample 5,830,741 5 6 Dilution Buffer Was added to the dissolved substrate to from the substrate FITC-casein according to a modi?ed a ?nal volume so that the concentration of substrate version of the method of TWining (Anal. Biochem. 1984; Was 1 mg/ml. 143:30). Fluorescent peptides Were quanti?ed using an 100 mM/l Calcium Chloride (CaCl2): 1.47 g CaCl2, excitation Wavelength of 491 nm and an emission Wave Formula Weight 147, Was dissolved in DI Water. length of 525 nm. The range of measured FITC-casein The volume Was adjusted to 100 ml With DI Water. speci?c activity for the neutral protease dispase Was typi 25 mM/l Citric Acid: 0.525 g Citric acid, FW 210.1, Was cally 600 to 1400 U/mg and Was more typically 1100 to 1400 dissolved in DI Water. The volume Was adjusted to 100 U/mg. The range of measured FITC-casein speci?c activity ml With DI Water. for the neutral protease thermolysin Was typically 3000 to 6500 U/mg and Was more typically 3500 to 6000 U/mg. One Assay Mixture: 2.0 ml of PZ-Pro-Leu-Gly-Pro-D-Arg 10 Substrate and 0.4 ml 100 mM CaCl2 Were added to unit of activity generates 100,000 ?uorescence units (counts 12x75 millimeter test tubes and capped. The per second) corrected for background per minute at 37° C., pH 7.5. A description of the test procedure used for the assay mixtures Were alloWed to equilibrate in a Water bath to 25° C. before beginning the assay. One tube Was neutral protease activity assay is given beloW. Sample Dilution Buffer (100 mM/l Tris, 10 mM/l CaCl2, prepared for each duplicate blank or sample tested. 15 Extraction Mixture: 5.0 ml of ethyl acetate and 1.0 ml of pH 7.5): 6.06 g Tris and 0.74 g CaCl2 Were dissolved in DI Water. The pH Was adjusted to 7.5 With 1.0N HCl 25 mM/l citric acid Were added to 16><125 mm test and the volume adjusted to 500 ml With DI Water. tubes. Each tube Was capped. One tube Was prepared for each duplicate blank or sample tested. FITC-Casein Substrate Solution (0.25% W/v): 50.0 mg FITC-casein Was dissolved in the Sample Dilution Drying tubes: 0.35 to 0.4 g anhydrous sodium sulfate Was 20 Buffer. The volume Was adjusted to 20.0 ml With added to 16><125 mm test tubes. Each tube Was capped. Sample Dilution Buffer. One tube Was prepared for each duplicate blank or sample tested. Quenching Solution (5.0% W/v): 5 .0 g of Trichloroacetic Assay Procedure: All concentrated and diluted enZyme Acid Was dissolved in DI Water. The volume Was adjusted to 100 ml With DI Water. samples Were kept at 2° C. to 8° C. until added to the 25 substrate. A sample of either the entire enZyme com Neutralization Solution (500 mM/l Tris: pH 8.5): 30.3 g position or one of its components (e.g., collagenase I, Tris Was dissolved in DI Water. The pH Was adjusted to collagenase II, or neutral protease) Was diluted With 8.5 With 1.0N HCl and the volume Was adjusted to 500 Sample Dilution Buffer to a concentration range of 0.05 ml With DI Water. to 0.8 mg/ml, depending upon estimated sample activ 30 Assay Procedure: All concentrated and diluted enZyme ity. The assay incubation time Was started after 100 samples Were kept at 2° C. to 8° C. until added to the microliters (,ul) of blank control (Sample Dilution substrate. Each blank or test sample Was assayed in Buffer Was used as a blank) Was added to the ?rst tube duplicate. A sample of either the entire enZyme com of Assay Mixture. The Assay Mixture Was then capped, position or one of its components (e.g., collagenase I, mixed thoroughly, and placed in a 25 ° C. Water bath. At 35 collagenase II, or neutral protease) Was diluted With 60 second intervals, 100 pl of the next blank or test Sample Dilution Buffer to a concentration range of 1 to sample Was added to a tube of Assay Mixture in the 50 micrograms (ug)/ml, depending upon estimated same manner. Each Assay Mixture Was incubated at sample activity. 10 pl of the diluted samples Were added 25° C. for 15 minutes (min) from the time the blank or to 40 pl of FITC-casein Substrate Solution in 1.5 ml test sample Was added. After 15 min of incubation, 0.5 40 Eppendorf tubes (the Sample Dilution Buffer Was used ml of the ?rst blank Assay Mixture Was transferred to as a blank control) and incubated for 45 min With a tube containing Extraction Mixture. The Extraction shaking at 37° C. in a Water bath. The tubes Were Mixture tube Was capped and mixed thoroughly by removed from the Water bath and 120 pl of the Quench vortexing for 20 seconds. At 60 second intervals, the ing Solution Was added. The tubes Were vortexed and remaining blank or test samples Were transferred to 45 left at room temperature for at least 60 min. The tubes Extraction Mixture tubes and mixed in the same Were then centrifuged in a microcentrifuge at 14,000 manner, so that total incubation time for each sample rpm for 2 min. 50 pl of the supernatant Was removed Was 15 min. 3 ml of the organic phase (top) from each and added to 2 ml of NeutraliZation Buffer in a cuvette. Extraction Mixture Was pipetted into a Drying Tube. The cuvette Was capped and inverted to mix the solu The Drying Tubes Were capped, mixed and incubated at tion. Fluorescence Was then measured (excitation room temperature in a fume hood for 30 to 60 min. The Wavelength 491 nm, emission Wavelength 525 nm, slit tubes Were mixed again during this period. The organic Width 0.2 nm). phase Was pipetted into quartZ cuvettes and, using a Calculations: CPS=Average enZyme sample temperature-controlled spectrophotometer at 25° C., ?uorescence-Average buffer blank ?uorescence. the absorbance Was read at 320 nanometers (nm) for 55 Activity (U/ml)=(CPS><0.17 ml>