ASTM D3048-89(2009)

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 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 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.

General Information

• Status: Historical
• Publication Date: 30-Sep-2009
• ICS: 71.100.40 - Surface active agents
• Technical Committee: D12 - Soaps and Other Detergents
• Drafting Committee: D12.12 - Analysis and Specifications of Soaps, Synthetics, Detergents and their Components

Relations

• Replaces: ASTM D3048-89(2003) - Standard Test Method of Assay for Alkaline Protease
• Effective Date: 01-Oct-2009
• Replaced By: ASTM D3048-89(2016) - Standard Test Method of Assay for Alkaline Protease
• Effective Date: 01-Jul-2016
• Referred By: ASTM D8179-18 - Standard Guide for Characterizing Detergents for the Cleaning of Clinically-used Medical Devices
• Effective Date: 01-Oct-2009

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D3048 − 89(Reapproved2009)
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 3.1.2 standardized enzyme—an enzyme preparation of
known activity for calibrating the sample enzyme in terms of a
1.1 This test method covers the assay of alkaline protease
3,4
gravimetric standard of enzymatic activity.
enzymes. This procedure is applicable to enzyme preparations
3.1.3 The terms “alkyl benzene sulfonate (ABS)” and “lin-
with high activity but is inapplicable to formulated detergent
ear alkylate sulfonate (LAS)” in this method are defined in
products or air samples.
accordance with Terminologies D1129 and D459:
3.1.3.1 alkyl benzene sulfonate (ABS)—the generic name
1.2 The values stated in SI units are to be regarded as
applied to the neutralized product resulting from the sulfona-
standard. No other units of measurement are included in this
tion of an alkylated benzene.
standard.
3.1.3.2 linear alkylate sulfonate (LAS)—a form of alkyl
1.3 This standard does not purport to address all of the
benzene sulfonate (ABS) in which the alkyl group is linear
safety concerns, if any, associated with its use. It is the rather than a branched chain.
responsibility of the user of this standard to establish appro- 3.1.4 nonionic surfactant—a mixed C -C linear primary
16 18
alcohol containing 65 % ethylene oxide.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Material Safety
3.1.5 For definitions of other terms used in these methods,
Data Sheets are available for reagents and materials. Review
refer to Terminology E131.
them for hazards prior to usage.
4. Summary of Test Method
2. Referenced Documents
4.1 This test is based on the hydrolysis of casein at 50°C for
15 min at pH 9. The trichloroacetic acid-soluble hydrolysate is
2.1 ASTM Standards:
assayed by the spectrophotometric determination of the absor-
D459 Terminology Relating to Soaps and Other Detergents
4,5
bance at approximately 275 nm. The results are correlated
D1129 Terminology Relating to Water
with the absorptivity of tyrosine or the absorbance of hydro-
D1193 Specification for Reagent Water
lysate from standardized enzyme. Results are reported asAPB,
E131 Terminology Relating to Molecular Spectroscopy
which is defined in Section 3, or in micrograms of pure
crystalline enzyme per gram of sample.
3. Terminology
5. Apparatus
3.1 Definitions:
3.1.1 APB unit—that amount of enzyme which releases in 1
5.1 Water Bath, constant-temperature, maintained at 50 6
min under the conditions of the test a casein hydrolysate that 0.2°C.
has the same absorbance as 1 µg of tyrosine in an equivalent
5.2 Ultraviolet Spectrophotometer, suitable for liquid mea-
volume. The number ofAPB units per gram of a preparation is
surements at a wavelength of approximately 275 nm.
called the APB of the preparation.
5.3 Absorption Cell, silica, 10-mm light path.
5.4 pH Meter.
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 and Specifications of Soaps, Synthetics, Detergents and their Components. The sole source of supply known to the committee at this time is National
Current edition approved Oct. 1, 2009. Published December 2009. Originally Institute of Occupational Safety and Health, 1014 BroadwayAve., Cincinnati, Ohio
approved in 1972 as D3048 – 72 T. Last previous edition approved in 2003 as 45202.
D3048 – 89 (2003). DOI: 10.1520/D3048-89R09. If you are aware of alternative suppliers, please provide this information to
For referenced ASTM standards, visit the ASTM website, www.astm.org, or ASTM International Headquarters. Your comments will receive careful consider-
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM ation at a meeting of the responsible technical committee, which you may attend.
Standards volume information, refer to the standard’s Document Summary page on Bailey, J. L. “Techniques in Protein Chemistry,” Elsevier Publishing Co., New
the ASTM website. York, NY. Chapter 11, 1967, pp. 340–352.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D3048 − 89(2009)
4,9
5.5 Test Tubes, 25 by 150 mm. Add 0.30 g of LAS (3.1.3) and 1.5 g of sodium tripolyphos-
phate. Stir to dissolve, and adjust to pH 9.0 with about 6 mLof
6. Reagents
0.1 N HCl. Dilute to 500 mL with water.
6.1 Purity of Reagent—Reagent grade chemicals shall be
6.11 TrichloroaceticAcid(TCA) Solution—Dissolve20.45g
used in all tests. Unless otherwise indicated, it is intended that
of TCA(CCl COOH) and 21.6 g of crystalline sodium acetate
all reagents shall conform to the specifications of the Commit-
trihydrate (CH COONa·3H O) in about 300 mL of water.Add
3 2
tee onAnalytical Reagents of theAmerican Chemical Society,
56.9 mL of 6.67 M CH COOH and dilute to 1 L with water.
where such specifications are available. Other grades may be
This solution is unstable and should be discarded after 1 week.
used, provided it is first ascertained that the reagent is of
6.12 Tyrosine Standard—Dissolve 100 mg of L-tyrosine,
sufficiently high purity to permit its use without lessening the
previously dried in a desiccator, in 60 mL of 0.1 N HCl. Upon
accuracy of the determination.
complete dissolution of the tyrosine dilute to 1 L with water.
6.2 Unless otherwise indicated, references to water shall be
understood to mean reagent water conforming to Specification 7. Safety Precautions
D1193.
7.1 Avoid generating or breathing enzyme dust.
6.3 Acetic Acid (6.67 M)—Mix 400 g of glacial acetic acid
8. Assay
(CH COOH) with sufficient water to yield 1 L of solution.
8.1 Sample—Prepare all solutions and serial dilutions of the
6.4 Borate Buffer (0.2 M)—Dissolve 12.4 g of boric acid
sample with enzyme buffer solution (6.5). Stock solutions
(H BO ) in 100 mL of 1 N NaOH solution and dilute to 1 L
3 3
should be stirred for 30 min before serial dilutions are made
with water.
and may be held for a maximum of 8 h. Use at least a 100 mg
6.5 Enzyme Buffer Solution—Dissolve 12.0 g of sodium
of the sample enzyme preparation for the initial stock solution.
chloride (NaCl) in about 500 mL of water and add 237 mL of
The final or working sample solution should contain 0.030 to
0.2 M borate buffer. Adjust to pH 9.0 with 0.1 N NaOH
0.060 mg/mL of solution. An activity of approximately
solution. Approximately 18 mL will be needed. Dilute to 1 L
600 000APBoranabsorbanceofapp
...