1、Designation: D 3048 89 (Reapproved 2003)Standard Test Method ofAssay for Alkaline Protease1This standard is issued under the fixed designation D 3048; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A num
2、ber in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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
3、is inapplicable to formulated detergentproducts or air samples.1.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applic
4、a-bility of regulatory limitations prior to use. Material SafetyData Sheets are available for reagents and materials. Reviewthem for hazards prior to usage.2. Referenced Documents2.1 ASTM Standards:D 459 Terminology Relating to Soaps and Other Deter-gents2D 1129 Terminology Relating to Water3D 1193
5、Specification for Reagent Water3E 131 Terminology Relating to Molecular Spectroscopy43. Terminology3.1 Definitions:3.1.1 APB unitthat amount of enzyme which releases in 1min under the conditions of the test a casein hydrolysate thathas the same absorbance as 1 g of tyrosine in an equivalentvolume. T
6、he 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 terms of agravimetric standard of enzymatic activity.53.1.3 The terms “alkyl benzene sulfonate (ABS)” and “lin-ear
7、 alkylate sulfonate (LAS)” in this method are defined inaccordance with Terminologies D 1129 and D 459:3.1.3.1 alkyl benzene sulfonate (ABS)the generic nameapplied to the neutralized product resulting from the sulfona-tion of an alkylated benzene.3.1.3.2 linear alkylate sulfonate (LAS)a form of alky
8、lbenzene 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 For definitions of other terms used in these methods,refer to Terminology E 131.4. Summary of Test Method4.1 This
9、 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 approximately 275 nm.6The results are correlatedwith the absorptivity of tyrosine or the absorbance of hydroly-sat
10、e 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-temperature, maintained at 50 60.2C.5.2 Ultraviolet Spectrophotometer, suitable for liquid mea-surements at a wave
11、length 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.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
12、 the Commit-tee on Analytical Reagents of the American Chemical Society,where such specifications are available.7Other grades may be1This test method is under the jurisdiction of ASTM Committee D12 on Soapsand Other Detergents and is the direct responsibility of Subcommittee D12.12 onAnalysis of Soa
13、ps and Synthetic Detergents.Current edition approved May 26, 1989. Published July 1989. Originallypublished as D 3048 72 T. Last previous edition D 3048 81 (1987).2Annual Book of ASTM Standards, Vol 15.04.3Annual Book of ASTM Standards, Vol 11.01.4Annual Book of ASTM Standards, Vol 03.06.5Available
14、from National Institute of Occupational Safety and Health, 1014Broadway Ave., Cincinnati, Ohio 45202.6Bailey, J. L. “Techniques in Protein Chemistry,” Elsevier Publishing Co., NewYork, NY. Chapter 11, 1967, pp. 340352.7Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Soc
15、iety, 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 States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD
16、.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.used, 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 indicate
17、d, references to water shall beunderstood to mean reagent water conforming to SpecificationD 1193.6.3 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 solut
18、ion and dilute to 1 Lwith water.6.5 Enzyme Buffer SolutionDissolve 12.0 g of sodiumchloride (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 volume
19、s of substrate(6.9) and synthetic detergent base (6.10) in sufficient quantityto 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
20、 Solution (1 N)Dissolve40 g of sodium hydroxide solution (NaOH) in water and diluteto1L.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 preparation5(3.1.2) in enzyme buffer solution(6.5), and dilute to 100 mL w
21、ith the same solution.6.9 SubstrateSlurry 6.0 g (dry basis) of casein8in 200 mLof water, add 120 mL of 0.2 M borate buffer, and heat 20 minin a boiling water bath. Cool to room temperature and adjust topH 9.0 with 0.1 N NaOH solution. About 30 mL will beneeded. Check the pH at higher dilution before
22、 adjusting withwater to a final volume of 500 mL. This solution is stable for1 week but should be stored under refrigeration.6.10 Synthetic Detergent BaseDissolve 0.3 g of nonionicsurfactant9(3.1.4) in 350 mL of water at 50C by stirring. Add0.30 g of LAS10(3.1.3) and 1.5 g of sodium tripolyphosphate
23、.Stir to dissolve, and adjust to pH 9.0 with about 6 mL of 0.1 NHCl. 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 dilu
24、te to 1 Lwith water. This solution is unstable and should be discardedafter 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
25、or breathing enzyme dust.8. Assay8.1 SamplePrepare all solutions and 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 preparatio
26、n for the initial stock solution.The final or working sample solution should contain 0.030 to0.060 mg/mL of solution. An activity of approximately600 000 APB or an absorbance of approximately 0.6 should beobserved for the 5-mL aliquot of sample solution used in thisassay.8.2 DigestionTriplicate anal
27、yses of samples and con-trolsare recommended.8.2.1 SampleThermally equilibrate 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 TCA reagent (6.11), shake vigorously, andretain at 50C for 30 min with
28、intermittent shaking.8.2.2 ControlIncubate 10 mL of enzyme media (6.6) andapproximately 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 vigoro
29、usly,and hold for 30 min as above.8.3 Removal of Precipitate:8.3.1 CentrifugationThe precipitated mixtures (8.2.1 and8.2.2) can be clarified by centrifugation.8.3.2 FiltrationFilter11the precipitated mixture after thor-ough shaking. Refilter the first portion of the filtrate throughthe same filter f
30、or a clear filtrate.8.4 Absorbance MeasurementDetermine the absorbanceof 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 determi
31、ne the absorbance of each at275 nm using a 10-mm cell path. Use a water blank to adjustthe instrument at zero absorbance. Prepare a graph of absor-bance versus g/mL of tyrosine for the range 25 to 100 g/mL.A straight line passing through the origin must be obtained. Anabsorptivity value of approxima
32、tely 0.0072 mL/(g cm) shouldresult.aT5 A/bc (1)where:aT= absorptivity value of tyrosine,A = absorbance of sample,b = cell path, 1 cm, andc = concentration, g/mL of tyrosine.8Hammersten casein is available from Nutritional Biochemical Corp. Cleveland,Ohio 44128.9Alfol 161865 available from Continenta
33、l Oil Co., Ponca City, OK 74601.10Sulframin 1345 available from Witco Chemical Corp., New York, NY. 1001shas been found suitable. The latter material is only 82.6 percent active and a suitableincrease must be made. LAS reference material may be obtained from the Soap alkaline protease; enzymeASTM In
34、ternational 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 of any such patent rights, and the riskof infringement of such rights, are entire
35、ly 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 are invited either for revision of this standard or for additional standardsand s
36、hould be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on S
37、tandards, 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 copies) 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).D 3048 89 (2003)3
