1、Designation: D3048 89 (Reapproved 2009)Standard Test Method ofAssay for Alkaline Protease1This standard is issued under the fixed designation D3048; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A numbe
2、r in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the assay of alkaline proteaseenzymes. This procedure is applicable to enzyme preparationswith high activity but is
3、inapplicable to formulated detergentproducts or air samples.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is ther
4、esponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Material SafetyData Sheets are available for reagents and materials. Reviewthem for hazards prior to usage.2. Referenced Documents
5、2.1 ASTM Standards:2D459 Terminology Relating to Soaps and Other DetergentsD1129 Terminology Relating to WaterD1193 Specification for Reagent WaterE131 Terminology Relating to Molecular Spectroscopy3. Terminology3.1 Definitions:3.1.1 APB unitthat amount of enzyme which releases in 1min under the con
6、ditions of the test a casein hydrolysate thathas the same absorbance as 1 g of tyrosine in an equivalentvolume. The number of APB units per gram of a preparation iscalled the APB of the preparation.3.1.2 standardized enzymean enzyme preparation ofknown activity for calibrating the sample enzyme in t
7、erms of agravimetric standard of enzymatic activity.3,43.1.3 The terms “alkyl benzene sulfonate (ABS)” and “lin-ear alkylate sulfonate (LAS)” in this method are defined inaccordance with Terminologies D1129 and D459:3.1.3.1 alkyl benzene sulfonate (ABS)the generic nameapplied to the neutralized prod
8、uct resulting from the sulfona-tion of an alkylated benzene.3.1.3.2 linear alkylate sulfonate (LAS)a form of alkylbenzene sulfonate (ABS) in which the alkyl group is linearrather than a branched chain.3.1.4 nonionic surfactanta mixed C16-C18linear primaryalcohol containing 65 % ethylene oxide.3.1.5
9、For definitions of other terms used in these methods,refer to Terminology E131.4. Summary of Test Method4.1 This test is based on the hydrolysis of casein at 50C for15 min at pH 9. The trichloroacetic acid-soluble hydrolysate isassayed by the spectrophotometric determination of the absor-bance at ap
10、proximately 275 nm.4,5The results are correlatedwith the absorptivity of tyrosine or the absorbance of hydroly-sate from standardized enzyme. Results are reported as APB,which is defined in Section 3, or in micrograms of purecrystalline enzyme per gram of sample.5. Apparatus5.1 Water Bath, constant-
11、temperature, maintained at 50 60.2C.5.2 Ultraviolet Spectrophotometer, suitable for liquid mea-surements at a wavelength of approximately 275 nm.5.3 Absorption Cell, silica, 10-mm light path.5.4 pH Meter.5.5 Test Tubes, 25 by 150 mm.1This test method is under the jurisdiction of ASTM Committee D12 o
12、n Soapsand Other Detergents and is the direct responsibility of Subcommittee D12.12 onAnalysis and Specifications of Soaps, Synthetics, Detergents and their Components.Current edition approved Oct. 1, 2009. Published December 2009. Originallyapproved in 1972 as D3048 72 T. Last previous edition appr
13、oved in 2003 asD3048 89 (2003). DOI: 10.1520/D3048-89R09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The
14、 sole source of supply known to the committee at this time is NationalInstitute of Occupational Safety and Health, 1014 Broadway Ave., Cincinnati, Ohio45202.4If you are aware of alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive caref
15、ul consider-ation at a meeting of the responsible technical committee,1which you may attend.5Bailey, J. L. “Techniques in Protein Chemistry,” Elsevier Publishing Co., NewYork, NY. Chapter 11, 1967, pp. 340352.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19
16、428-2959, United States.6. Reagents6.1 Purity of ReagentReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where such specificatio
17、ns are available.6Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.6.2 Unless otherwise indicated, references to water shall beunderstood to mean reagent water conforming to S
18、pecificationD1193.6.3 Acetic Acid (6.67 M)Mix 400 g of glacial acetic acid(CH3COOH) with sufficient water to yield 1 L of solution.6.4 Borate Buffer (0.2 M)Dissolve 12.4 g of boric acid(H3BO3) in 100 mL of 1 N NaOH solution and dilute to 1 Lwith water.6.5 Enzyme Buffer SolutionDissolve 12.0 g of sod
19、iumchloride (NaCl) in about 500 mL of water and add 237 mL of0.2 M borate buffer. Adjust to pH 9.0 with 0.1 N NaOHsolution. Approximately 18 mL will be needed. Dilute to 1 Lwith water.6.6 Enzyme MediaCombine equal volumes of substrate(6.9) and synthetic detergent base (6.10) in sufficient quantityto
20、 accommodate each sample and thermally equilibrate thissolution at 50C. Each analysis requires 20 mL of this solution,10 for assay and 10 for the control; samples should be run intriplicate.6.7 Sodium Hydroxide, Standard Solution (1 N)Dissolve40 g of sodium hydroxide solution (NaOH) in water and dil
21、uteto1L.Fora0.1N solution, dilute 100 mL of 1 N NaOHsolution to 1 L with water.6.8 Standardized EnzymeDissolve 100 mg of the stan-dardized enzyme preparation4,3(3.1.2) in enzyme buffer solu-tion (6.5), and dilute to 100 mL with the same solution.6.9 SubstrateSlurry 6.0 g (dry basis) of casein4,7in 2
22、00mL of water, add 120 mL of 0.2 M borate buffer, and heat 20min in a boiling water bath. Cool to room temperature andadjust to pH 9.0 with 0.1 N NaOH solution. About 30 mL willbe needed. Check the pH at higher dilution before adjustingwith water to a final volume of 500 mL. This solution is stablef
23、or 1 week but should be stored under refrigeration.6.10 Synthetic Detergent BaseDissolve 0.3 g of nonionicsurfactant4,8(3.1.4) in 350 mL of water at 50C by stirring.Add 0.30 g of LAS4,9(3.1.3) and 1.5 g of sodium tripolyphos-phate. Stir to dissolve, and adjust to pH 9.0 with about 6 mL of0.1 N HCl.
24、Dilute to 500 mL with water.6.11 Trichloroacetic Acid (TCA) SolutionDissolve 20.45g of TCA (CCl3COOH) and 21.6 g of crystalline sodiumacetate trihydrate (CH3COONa3H2O) in about 300 mL ofwater. Add 56.9 mL of 6.67 M CH3COOH and dilute to 1 Lwith water. This solution is unstable and should be discarde
25、dafter 1 week.6.12 Tyrosine StandardDissolve 100 mg of L-tyrosine,previously dried in a desiccator, in 60 mL of 0.1 N HCl. Uponcomplete dissolution of the tyrosine dilute to 1 L with water.7. Safety Precautions7.1 Avoid generating or breathing enzyme dust.8. Assay8.1 SamplePrepare all solutions and
26、serial dilutions of thesample with enzyme buffer solution (6.5). Stock solutionsshould be stirred for 30 min before serial dilutions are madeand may be held for a maximum of 8 h. Use at least a 100 mgof the sample enzyme preparation for the initial stock solution.The final or working sample solution
27、 should contain 0.030 to0.060 mg/mL of solution. An activity of approximately600 000APB or an absorbance of approximately 0.6 should beobserved for the 5-mL aliquot of sample solution used in thisassay.8.2 DigestionTriplicate analyses of samples and con-trolsare recommended.8.2.1 SampleThermally equ
28、ilibrate 5-mL aliquots ofsample solution (8.1) in 25 by 150-mm tubes. Add 10 mL ofenzyme media (6.6) and digest for 15 min in a water bath at50C.Add 10 mL of TCAreagent (6.11), shake vigorously, andretain at 50C for 30 min with intermittent shaking.8.2.2 ControlIncubate 10 mL of enzyme media (6.6) a
29、ndapproximately 5 mL of sample solution (8.1) for 15 min at50C in separate tubes. Add 10 mL of TCA reagent (6.11)tothe 10-mL aliquot of enzyme media, shake, and hold 1 min.Add 5 mL of the incubated sample solution, shake vigorously,and hold for 30 min as above.8.3 Removal of Precipitate:8.3.1 Centri
30、fugationThe precipitated mixtures (8.2.1 and8.2.2) can be clarified by centrifugation.8.3.2 FiltrationFilter4,10the precipitated mixture afterthorough shaking. Refilter the first portion of the filtratethrough the same filter for a clear filtrate.8.4 Absorbance MeasurementDetermine the absorbanceof
31、the supernatant in a 10-mm cell at 275 nm. Set theinstrument at zero absorbance with the incubated controlsolution (8.2.2).9. Calibration9.1 Tyrosine StandardPrepare serial dilutions of the ty-rosine standard (6.12) and determine the absorbance of each at275 nm using a 10-mm cell path. Use a water b
32、lank to adjust6Reagent Chemicals, American Chemical Society Specifications , AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United St
33、ates Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.7The sole source of supply of Hammersten casein known to the committee at thistime is Nutritional Biochemical Corp. Cleveland, Ohio 44128.8The sole source of supply of the Alfol 161865 known to the comm
34、ittee at thistime is Continental Oil Co., Ponca City, OK 74601.9The sole source of supply of Sulframin 1345 known to the committee at thistime is Witco Chemical Corp., New York, NY. 1001s has been found suitable. Thelatter material is only 82.6 percent active and a suitable increase must be made. LA
35、Sreference material may be obtained from the Soap alkaline protease; enzymeASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity
36、 of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments
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38、received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copie
39、s) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).D3048 89 (2009)3
