Datasheet - Sigma

Document technical information

Format pdf
Size 88.9 kB
First found May 22, 2018

Document content analysis

Category Also themed
Language
English
Type
not defined
Concepts
no text concepts found

Persons

Organizations

Places

Transcript

Lysozyme
from chicken egg white
for Molecular Biology
Catalog Number L7651
Storage Temperature –20 °C
CAS RN 12650-88-3
EC 3.2.1.17
Synonyms: Muramidase; Lysozyme c;
Mucopeptide N-acetylmuramoylhydrolase
Product Description
Lysozyme is a single chain polypeptide of 129 amino
acids cross-linked with four disulfide bridges.1 It
hydrolyzes β(1→4) linkages between N-acetylmuraminic acid and N-acetyl-D-glucosamine residues in
peptidoglycan and between N-acetyl-D-glucosamine
residues in chitodextrin.2,3 The enzyme is often used for
lysing bacterial cells by hydrolyzing the peptidoglycan
present in the cell walls. Gram-positive cells are quite
susceptible to this hydrolysis as their cell walls have a
high proportion of peptidoglycan. Gram-negative
bacteria are less susceptible due to the presence of an
outer membrane and a lower proportion of
peptidoglycan. However, these cells may be hydrolyzed
more easily in the presence of EDTA that chelates
metal ions in the outer bacterial membrane.4,5
This lysozyme preparation is purified from chicken egg
white, crystallized three times, dialyzed, and supplied
as a lyophilized powder. Protein content by UV
absorbance is ≥90% with the remainder (∼10%) being
buffer salts such as sodium acetate and sodium
chloride.
This highly purified enzyme preparation has been used
in mass spectrometry as a protein mass calibration
standard and in structural studies of proteins.6-8 It is
suitable for use as a lysing agent in the purification of
9
plasmid DNA using a boiling lysing technique.
Molecular mass:10 14,307 Da (amino acid sequence)
Isoelectric point (pI):
11
11.35
Extinction co-efficients:
E1%(281.5 nm):12 26.4 in 0.1 M potassium chloride
EmM (280 nm):13 36
Optimal pH:
The activity of lysozyme is a function of both pH and
ionic strength. The enzyme is active over a broad pH
range (6.0–9.0). At pH 6.2, maximal activity is observed
over a wider range of ionic strengths (0.02–0.100 M)
than at pH 9.2 (0.01–0.06 M).13
Inhibitors:
Lysozyme is inhibited by indole derivatives, which bind
to and distort the active site, and imidazole, which
induces the formation of a charge-transfer complex.14
It is also inhibited by surface-active agents such as
sodium dodecyl sulfate, sodium dodecanate, and
dodecyl alcohol. Other compounds of these types with
carbon chains of 12 or more carbons in length will also
inhibit lysozyme.15
Substrates:
The natural substrate for lysozyme is the peptidoglycan
layer of bacterial cell walls. However, a variety of low
molecular mass substrates including murein
degradation products as well as synthetic compounds
have been used for various photometric, isotopic, and
immunological lysozyme assays.16
The following low molecular mass lysozyme substrates
are available:
4-Methylumbelliferyl β-D-N,N′,N′′-triacetyl-chitotrioside
(Catalog Number M5639, a fluorogenic substrate)
4-Nitrophenyl β-D-N,N′,N′′-triacetylchitotriose
(Catalog Number N8638)
Lysozyme activity: ≥40,000 units/mg protein
Unit definition: One unit will produce a change in A450 of
0.001 per minute at pH 6.24 at 25 °C, using a
suspension of Micrococcus lysodeikticus as substrate,
in a 2.6 ml reaction mixture (1 cm light path).
Precautions and Disclaimer
This product is for R&D use only, not for drug,
household, or other uses. Please consult the Material
Safety Data Sheet for information regarding hazards
and safe handling practices.
Preparation Instructions
For E. coli cell lysis, use a freshly prepared lysozyme
9
solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.
The product is also soluble in water (10 mg/ml) yielding
a clear to slightly hazy colorless solution. Aqueous
solutions should retain activity for at least one month
when stored between 2–8 °C.
Storage/Stability
The product, as supplied, should be stored at –20 °C.
When stored at –20 °C, the enzyme retains activity for
at least 4 years.
Solutions (pH 4–5) remain active for several weeks if
refrigerated.
Procedure
The following procedure is for the lysis of E. coli. It may
be used as a guideline for other species. The optimal
pH for E. coli cell lysis is 8.0±0.1.9
1. Incubate E. coli (strain ATCC 37017) bearing the
pBR322 plasmid overnight in Terrific Broth (Catalog
Number T0918) with 25 µg/ml tetracycline (Catalog
Number T3383) and 25 µg/ml ampicillin (Catalog
Number A9518).
2. Centrifuge 1–2 ml samples of the overnight culture.
3. Resuspend the pellets in 350 µl of STET buffer
(10 mM Tris-HCl, pH 8.0, with 0.1 M NaCl, 1 mM
EDTA, and 5% [w/v] TRITON X-100).
4. Add 25 µl of a freshly prepared lysozyme solution
(10 mg/ml in 10 mM Tris-HCl, pH 8.0).
5. Mix by vortexing for 3 seconds.
6. Incubate the lysis mixture for 30 minutes at 37 °C
7. After incubation, place the tube containing the lysis
mixture in a boiling water bath for exactly
40 seconds.
8. Centrifuge the lysis mixture at 14,000 × g.
9. Remove the pellet (cell debris) from the tube using
a sterile toothpick.
10. Plasmid DNA from the supernatant may then be
purified and analyzed.
References
1. Jolles, P., Angewandte Chemie, International
Edition, 8, 227-239 (1969).
2. Rupley, J.A., Biochim. Biophys. Acta, 83, 245-255
(1964).
3. Holler, H., et al., Biochem., 14, 2377-2385 (1975).
4. Schutte, H., et al., Biotech. Applied Biochem., 12,
599-620 (1990).
5. Vazquez,-Laslop, N., et al., J. Bact., 183, 23992404 (2001).
6. Galvani, M., et al., Electrophoresis, 22, 2058-2065
(2001).
7. Abgar, S., et al., Eur. J. Biochem., 267, 5916-5925
(2000).
8. Sethuraman, A., et al., Proteins: Structure, Function
and Bioinformatics, 56, 669-678 (2004).
9. Sambrook, J., et al., in Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory
Press, (Cold Spring Harbor, NY: 1989) p 1.29.
10. Canfield, R.E., J. Biol. Chem., 238, 2698-2707
(1963).
11. Wetter, L.R., et al., J. Biol. Chem., 192, 237-242
(1951).
12. Aune, K.C., et al., Biochem., 8, 4579-4585 (1965).
13. Davies, R.C., et al., Biochim. Biophys. Acta, 178,
294-305 (1969).
14. Swan, I., J. Mol. Bio., 65, 59-62 (1972).
15. Smith, G., and Stoker, C., Arch. Biochem. Biophys.,
21, 383-394 (1949).
16. Holtje, J.V., EXS, 75, 105-110 (1996).
TRITON is a registered trademark of the Union Carbide
Corp.
CS,RBG,ALC,MAM 03/08-1
Sigma brand products are sold through Sigma-Aldrich, Inc.
Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser
must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of
the invoice or packing slip.

Similar documents

×

Report this document