Sol–gel encapsulation of acid phosphatase
123
Spectronic 20D digital UV–visible spectrophotometer. Our
blank, a control sample without APase, showed no mea-
surable increase in absorbance at 410 nm. To conduct
assays on sol–gel-encapsulated APase, four APase sol–gel
beads containing 0.45 mg APase in total were placed as
wet hydrogel beads in a test tube with 2.4 cm3 20 mM Tris
buffer, pH 7.2. The reaction was initiated by adding
0.6 cm3 50 mM stock solution of 1. The contents of the test
tube were gently mixed by inversion between the absor-
bance readings that were recorded every 30 s at 410 nm.
As control, we prepared sol–gel beads without APase.
A blank prepared with these empty beads showed no
measurable increase in absorbance at 410 nm. All samples
were prepared in triplicate. The standard deviation did not
exceed 20% of the mean value. APase activity is reported
in U/mg. One U/mg corresponds to the hydrolysis of
1 lmol p-nitrophenylphosphate/min by 1 mg APase.
All samples for the thermal deactivation study were
subjected to heat shock by placing them in a 60 °C water
bath for varying periods of time. The heat-shocked samples
were dipped into a 25 °C water bath for 5 min and kinetic
assays were conducted immediately thereafter at room
temperature.
1 mm thick and the cassette was sealed with parafilm. The
sol–gel solution was prepared by sonicating 7.37 cm3
TMOS, 0.92 cm3 dH2O, and 0.88 cm3 0.01 M hydrochlo-
ric acid for 20 min in an ice–water bath. The final protein
concentration after dilution with the sol–gel solution was
0.56 mg/cm3. A sample containing 0.56 mg/cm3 free
APase in 2 mM Tris buffer pH 7.2 was prepared from the
same APase stock solution and at the same time as the sol–
gel sample. Both samples were stored for 2 weeks at 4 °C.
Shortly before the CD measurement, the cassette housing
the APase sol–gel was opened. A small rectangle was cut
with a razor blade and transferred into a CD cuvette.
Gelation of the APase sol–gel resulted in a slightly shrunk
hydrogel sheet that easily fit into a CD cuvette 1 mm thick.
The same cuvette was used for the APase solution sample.
CD spectra were recorded with an AVIV Model 215
circular dichroism spectrometer in 1 nm increments.
The temperature was set to 25 °C and controlled with a
thermostat.
Acknowledgments The principal investigator (Monika Sommer-
halter) received a Faculty Research Grant and an Interdisciplinary
Sieber-Tombari Research Award from California State University,
East Bay. The first author (Suhasini Kulkarni) received a graduate
student research grant from California State University, East Bay. The
second author (Elizabeth Chin) gratefully acknowledges financial
support from the CSU-LSAMP program which is supported by the
National Science Foundation under Grant No. HRD-0331537. CD
measurements were performed in the laboratory of Professor Daryl
Eggers at San Jose State University. We thank Dr Eggers for his kind
hospitality and support during the measurements.
Fixed-time assays were used to determine the pH profile
of APase. Each test tube contained 500 mm3 buffer at a
given pH, 500 mm3 15.2 mM 1, and 100 mm3 APase sam-
ple. The amount of free APase was 0.05 mg per test tube and
the amount of sol–gel-encapsulated APase was 0.225 mg
distributed in two hydrogel beads each with a volume of
50 mm3. The buffers used were: 50 mM acetate, pH 4.0,
50 mM acetate, pH 5.0, 50 mM 2-(N-morpholino)ethane-
sulfonic acid, pH 6.0, 50 mM Tris, pH 7.0, 50 mM Tris, pH
8.0, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid, pH
9.0, and 50 mM 3-(N-cyclohexylamino)propanesulfonic
acid, pH 10.0. After 10 min, 4 cm3 100 mM NaOH was
added, and the absorbance at 410 nm was recorded. Under
these conditions (pH 12) the extinction coefficient of 2 at
References
1. Gill I (2001) Chem Mater 13:3404
2. Wei Y, Xu JG, Feng QW, Lin MD, Dong H, Zhang WJ, Wang C
(2001) J Nanosci Nanotechnol 1:83
3. O’Neill H, Angley CV, Hemery I, Evans BR, Dai S, Woodward J
(2002) Biotechnol Lett 24:783
4. Frenkel-Mullerad H, Avnir D (2005) J Am Chem Soc 127:8077
5. Kragl U, Eckstein M, Kaftzik N (2002) Curr Opin Biotech 13:565
6. Karout A, Pierre AC (2007) J Non-Cryst Solids 353:2900
7. Liu Y, Wang MJ, Li J, Li ZY, He P, Liu HT, Li JH (2005) Chem
Commun 1778
410 nm is 18.3 0.2 mM-1 cm-1
.
Circular dichroism
8. Lee SH, Doan TTN, Ha SH, Koo YM (2007) J Mol Catal B
Enzym 45:57
9. Joyce BK, Grisolia S (1960) J Biol Chem 235:2278
10. He ZQ, Griffin TS, Honeycutt CW (2004) J Environ Qual 33:367
11. Machado MF, Saraiva JM (2005) Biotechnol Lett 27:1233
To prepare an APase hydrogel sample that fits into a 1-mm
circular dichroism (CD) cuvette, 3.5 cm3 sol–gel solution
were gently mixed with 5.25 cm3 APase stock solution
containing 0.93 mg/cm3 APase in 2 mM Tris, pH 7.2. This
mixture was cast into a plastic cassette (Novex/Invitrogen)
123