ASTM D3048-89(2003)
(Test Method)Standard Test Method of Assay for Alkaline Protease
Standard Test Method of Assay for Alkaline Protease
ABSTRACT
This test method covers the assay of alkaline protease enzymes. This procedure is applicable to enzyme preparations with high activity but is inapplicable to formulated detergent products or air samples. The apparatus and reagents used in testing the assay for alkaline protease enzymes are presented.
SCOPE
1.1 This test method covers the assay of alkaline protease enzymes. This procedure is applicable to enzyme preparations with high activity but is inapplicable to formulated detergent products or air samples.
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Material Safety Data Sheets are available for reagents and materials. Review them for hazards prior to usage.
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Designation:D3048–89(Reapproved 2003)
Standard Test Method of
Assay for Alkaline Protease
This standard is issued under the fixed designation D3048; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.3.1 alkyl benzene sulfonate (ABS)—the generic name
applied to the neutralized product resulting from the sulfona-
1.1 This test method covers the assay of alkaline protease
tion of an alkylated benzene.
enzymes. This procedure is applicable to enzyme preparations
3.1.3.2 linear alkylate sulfonate (LAS)—a form of alkyl
with high activity but is inapplicable to formulated detergent
benzene sulfonate (ABS) in which the alkyl group is linear
products or air samples.
rather than a branched chain.
1.2 This standard does not purport to address all of the
3.1.4 nonionic surfactant—a mixed C -C linear primary
16 18
safety concerns, if any, associated with its use. It is the
alcohol containing 65 % ethylene oxide.
responsibility of the user of this standard to establish appro-
3.1.5 For definitions of other terms used in these methods,
priate safety and health practices and determine the applica-
refer to Terminology E131.
bility of regulatory limitations prior to use. Material Safety
Data Sheets are available for reagents and materials. Review
4. Summary of Test Method
them for hazards prior to usage.
4.1 This test is based on the hydrolysis of casein at 50°C for
2. Referenced Documents 15 min at pH 9. The trichloroacetic acid-soluble hydrolysate is
assayed by the spectrophotometric determination of the absor-
2.1 ASTM Standards:
bance at approximately 275 nm. The results are correlated
D459 Terminology Relating to Soaps and Other Detergents
with the absorptivity of tyrosine or the absorbance of hydroly-
sate from standardized enzyme. Results are reported as APB,
D1129 Terminology Relating to Water
which is defined in Section 3, or in micrograms of pure
D1193 Specification for Reagent Water
crystalline enzyme per gram of sample.
E131 Terminology Relating to Molecular Spectroscopy
5. Apparatus
3. Terminology
5.1 Water Bath, constant-temperature, maintained at 50 6
3.1 Definitions:
0.2°C.
3.1.1 APB unit—that amount of enzyme which releases in 1
5.2 Ultraviolet Spectrophotometer, suitable for liquid mea-
min under the conditions of the test a casein hydrolysate that
surements at a wavelength of approximately 275 nm.
has the same absorbance as 1 µg of tyrosine in an equivalent
5.3 Absorption Cell, silica, 10-mm light path.
volume. The number ofAPB units per gram of a preparation is
5.4 pH Meter.
called the APB of the preparation.
5.5 Test Tubes, 25 by 150 mm.
3.1.2 standardized enzyme—an enzyme preparation of
known activity for calibrating the sample enzyme in terms of a
6. Reagents
gravimetric standard of enzymatic activity.
6.1 Purity of Reagent—Reagent grade chemicals shall be
3.1.3 The terms “alkyl benzene sulfonate (ABS)” and “lin-
used in all tests. Unless otherwise indicated, it is intended that
ear alkylate sulfonate (LAS)” in this method are defined in
all reagents shall conform to the specifications of the Commit-
accordance with Terminologies D1129 and D459D1129D459:
tee onAnalytical Reagents of theAmerican Chemical Society,
where such specifications are available. Other grades may be
used, provided it is first ascertained that the reagent is of
This test method is under the jurisdiction of ASTM Committee D12 on Soaps
and Other Detergents and is the direct responsibility of Subcommittee D12.12 on
Analysis of Soaps and Synthetic Detergents.
Current edition approved May 26, 1989. Published July 1989. Originally Bailey, J. L. “Techniques in Protein Chemistry,” Elsevier Publishing Co., New
published as D3048 – 72 T. Last previous edition D3048 – 81 (1987). DOI: York, NY. Chapter 11, 1967, pp. 340–352.
10.1520/D3048-89R03. Reagent Chemicals, American Chemical Society Specifications, American
Annual Book of ASTM Standards, Vol 15.04. Chemical Society, Washington, DC. For suggestions on the testing of reagents not
Annual Book of ASTM Standards, Vol 11.01. listed by the American Chemical Society, see Analar Standards for Laboratory
Annual Book of ASTM Standards, Vol 03.06. Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Available from National Institute of Occupational Safety and Health, 1014 and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
Broadway Ave., Cincinnati, Ohio 45202. MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D3048–89 (2003)
sufficiently high purity to permit its use without lessening the 8. Assay
accuracy of the determination.
8.1 Sample—Prepare all solutions and serial dilutions of the
6.2 Unless otherwise indicated, references to water shall be
sample with enzyme buffer solution (6.5). Stock solutions
understood to mean reagent water conforming to Specification
should be stirred for 30 min before serial dilutions are made
D1193.
and may be held for a maximum of 8 h. Use at least a 100 mg
6.3 Acetic Acid (6.67 M)—Mix 400 g of glacial acetic acid
of the sample enzyme preparation for the initial stock solution.
(CH COOH) with sufficient water to yield 1 L of solution.
The final or working sample solution should contain 0.030 to
6.4 Borate Buffer (0.2 M)—Dissolve 12.4 g of boric acid
0.060 mg/mL of solution. An activity of approximately
(H BO ) in 100 mL of 1 N NaOH solution and dilute to 1 L
3 3
600 000APBoranabsorbanceofapproximately0.6shouldbe
with water.
observed for the 5-mL aliquot of sample solution used in this
6.5 Enzyme Buffer Solution—Dissolve 12.0 g of sodium
assay.
chloride (NaCl) in about 500 mL of water and add 237 mL of
8.2 Digestion—Triplicate analyses of samples and con-trols
0.2 M borate buffer. Adjust to pH 9.0 with 0.1 N NaOH
are recommended.
solution. Approximately 18 mL will be needed. Dilute to 1 L
8.2.1 Sample—Thermally equilibrate 5-mL aliquots of
with water.
sample solution (8.1) in 25 by 150-mm tubes. Add 10 mL of
6.6 Enzyme Media—Combine equal volumes of substrate
enzyme media (6.6) and digest for 15 min in a water bath at
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