Chapter 5 Decrease in size of hen egg white lysozyme aggregates with decrease in monomer concentration from micro to
2.2 Methods .1 Stock solutions
To investigate the hen lysozyme aggregation, concentration of stock solution of HEWL was measured using UV specrophotometric method. It is well known that lysozyme contains aromatic amino acids that absorb UV light at 280 nm. The concentration of HEWL stock solution (10 mg/ml) made in deionised water was verified using extinction coefficient 38,400 M-1cm-1 at 280 nm.
To investigate the effects of additive on aggregation, various additives were used such as surfactant (SDS, CTAB) and DTT. These additives 14 mM SDS, 3 mM CTAB and 20 mM DTT were prepared in sodium dihydrogen phosphate buffer, pH 12.2 from their respective stock solution. Samples containing DTT were kept sealed to prevent air oxidation.
For characterization of HEWL aggregates after prior incubation with chitotriose and NAG, the 50mM chitotriose and 250 mM NAG stock solution was prepared in deionised water (MilliQ).
For the free -SH estimation, stock solution of 60 mM L-Cysteine was prepared in deionised water (MilliQ) and 32 mM DTNP was prepared in DMSO, It was vortexed vigorously for complete dissolution, and Stock solution of DTNP was made freshly for every analysis.
2.2.2 Sample preparation for aggregation and control
A different kind of buffer was used to carry out the work as follows: 50 mM Sodium dihydrogen phosphate pH 7.0 for control and pH 12.2 for aggregation study, was prepared in deionised water (MilliQ). For dye labeling 100 mM sodium bicarbonate pH 9 and pH 8.3 was prepared in deionised water (MilliQ).
(A) Sample for aggregation: Diluting from stock solution (10 mg/ml), 120 µM HEWL sample was prepared in 50 mM phosphate buffer pH 12.2 and it was kept for aggregation at room temperature (25-28 °C). Precipitation was not seen while incubation at 25-28 °C for [HEWL
< 200µM]. Control sample 120 µM HEWL was prepared in 50 mM phosphate buffer pH 7.0, kept at room temperature (25-28 °C). The sample for 0.3 µM and 0.03 µM (300 &30 nM) was prepared by serial dilution from stock solution.
(B) Sample in presence of surfactants (SDS, CTAB) and DTT: Separately 14 mM SDS and 20 mM DTT from their respective stock solution (stock solution in 50 mM phosphate buffer pH 12.2) were added in 120 µM HEWL, desired volume was maintained from pH 12.2 buffer, whereas for 3 mM CTAB from stock solution (stock solution in 50 mM phosphate buffer pH 12.2) added in 80 µM HEWL sample instead of 120 µM HEWL concentration. At 120µM concentration precipitation was observed in CTAB containing sample. Samples containing
DTT were kept under sealed to prevent air oxidation. All experiments were performed using various biophysical techniques at 25-28 ºC.
(C) Sample in presence of chitotriose: 50 mM chitotriose stock solution was prepared in deionised water (MilliQ), immediately before starting the over night incubation in 10 mM pH 7.3 phosphate buffer. Next 5 mM chitotriose was added in 300 µM HEWL with rest of the volume maintained with 10 mM phosphate buffer pH 7.3. This sample was kept at room temperature overnight. Later it was diluted to 100 µM HEWL containing >1.5 mM chitotriose (from over night incubated sample) in pH 12.2 buffer, and kept for incubation to observe the effect on aggregation.
(D) Sample in presence of NAG: Stock solution of 250 mM NAG was prepared in deionised water (MilliQ), immediately before starting the over night incubation in 10 mM pH 7.3 phosphate buffer. Next 25 mM NAG was added in 300 µM HEWL with rest of the volume maintained with 10 mM phosphate buffer pH 7.3. After making the sample it was kept at room temperature overnight. Later it was diluted to 100 µM HEWL containing >7.5 mM NAG (from over night incubated sample) in pH 12.2 buffer, and kept for incubation to observe the effect on aggregation.
2.2.3 Labeling HEWL with dansyl and dabcyl probes
HEWL was covalently labeled with dansyl chloride following the protocol recommended by Molecular Probes with minor modification as reported previously (Homchaudhuri et al., 2006). HEWL was labeled with dansyl chloride follows:
(2-dimethyl aminonaphthalene-6-sulfonyl chloride)
Step 1: HEWL (12 mg) was dissolved in 1 ml of freshly prapred 100 mM sodium bicarbonate pH 9.0 buffer in a glass vial. Dansyl chloride (2-6 mg) was carefully dissolved in 100 µL of DMF in eppendroff tube. Dye stock solution wrapped with aluminium foil and direct light was avoided.
Step 2: Dissolved HEWL was stirred using rice magetic beads and 100 µL dansyl chloride added slowly. For the complete reaction sample was stirred continuously for 3 hrs at 4 °C in cold room.
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Reaction scheme 1: Adopted from Molecular Probes catalog for sulfonyl choride dye with HEWL.
Step 3: After completing the reaction, conjugated HEWL-dansyl was separated using PD10 column. (A) Before eluting the labeled sample, column was equilibrated with 25 ml of 50 mM phosphate pH 7.0 buffer. (B) Then labeled sample was diluted adding 1.5 ml of sodium bicarbonate pH 9.0 buffer, and final volume was 2.6 ml of labeled sample. (C) The 2.6 ml sample was added to column and the flow through was discarded. (D) After the discarding the flow through, 3.5 ml phosphate pH 7.0 buffer was added. (E) Finally four fraction of 1 ml each was collected and it was stored at 4 °C.
Step 4: The HEWL, dansyl concentrations were estimated by measuring the absorbance at 280 nm (ε = 38,400 M-1 cm-1), 380 nm (16,000 M-1cm-1) The protein to dye labeling ratio in the conjugate were consistently between 2-3.
For labeling HEWL with dabcyl, the protocol suggested by Molecular Probes was followed
(4-((4-(dimethylamino) phenyl) azo) benzoic acid, succinimidyl ester)
Step 1: HEWL (16 mg ) was dissolved in 1 ml of freshly prepared 100 mM sodium