ASTM_D_3048_-_89_2016.pdf

Designation:D304889(Reapproved 2016)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 number 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 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 theresponsibility 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 Documents2.1 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 conditions 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 terms 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 product 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 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 approximately 275 nm.4,5The results are correlatedwith the absorptivity of tyrosine or the absorbance of hydro-lysate 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 wavelength of approximately 275 nm.5.3 Absorption Cell,silica,10-mm light path.5.4 pH Meter.1This test method is under the jurisdiction of ASTM Committee D12 on 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 July 1,2016.Published August 2016.Originallyapproved in 1972 as D3048 72 T.Last previous edition approved in 2009 asD3048 89(2009).DOI:10.1520/D3048-89R16.2For 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 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 careful 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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 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 the Commit-tee on Analytical Reagents of the American Chemical Society,where such specifications 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