Ch3cooh

Preparation of 2L of of buffered acetic acid solution (Step 4):1.

Begin with 1L MQ water in a 2L bottle (MQ water = Milli-Q® purified water that has resistance of 18.2 MΩ•cm at 25°C)

2.

Add 164g sodium acetate (C2H3NaO2 Anhydrous, Fisher Chemical, Certified ACS, F.W. = 82.03, S210-2)

3.

Add 114mL glacial acetic acid (CH3COOH, Fisher Chemical, Certified ACS, F.W.=60.05, product A38C-212)

4.

Fill to 2L with MQ

Preparation of 500mL of buffered acetic acid/sodium acetate (pH 4) solution (Step 5):1.

start with 250mL MQ

2.

Add 1.84g hydroxylamine hydrochloride (H3NO•HCl, Alfa Aesar, 99% purity, F.W. 69.49, Stock #: A15398)

3.

Add 76mL glacial acetic acid (CH3COOH)

4.

Add 10g NaOH pellets (Fisher Chemical, Certified ACS, F.W. = 40, Catalogue #: S318-1)

5.

Fill to 500mL with MQ

6.

Check pH after 1hr; should be ~4

The recombinant azurin mutant Y72F/Y108F
from Pseudomonas aeruginosa was expressed and purified
as described previously^{9 (link),10 (link)} with modifications.^{13 (link)} The single-tryptophan apoprotein mutant was
generated from the holoprotein using a cyanide procedure^{29 (link)} and stored in 50 mM acetate buffer at pH 4.5.
The apoprotein is referred to as apoAzW48. The ratio of absorbance
at 630 to 280 nm of the purified apoprotein was less than 0.003. All
experiments were performed in 20 mM phosphate buffer at pH 7.3. An
aliquot of stock 10.0 mM aqueous solution of the exogenous electron
acceptor [Co(NH₃)₅Cl]²⁺ was added
to azurin samples when appropriate. N-Acetyl-l-tryptophanamide (NATA) was prepared as a 0.1 mM aqueous, buffered
(phosphate, pH 7.2) stock solution for fluorescence quantum yield
measurements. The reagents were obtained from the following commercial
sources and used without purification: K₂HPO₄ and KH₂PO₄ salts from Fisher Chemical; [Co(NH₃)₅Cl]Cl₂ (98%) from Sigma-Aldrich; KCN
(96%) from Spectrum Chemical; NaCH₃COO (99%) and CH₃COOH (99%) from Fisher Chemical; CuSO₄ (99%) from
Alfa Aesar; and NATA (98%) from Sigma-Aldrich.

Polysaccharides, pectin from citrus (TCI Europe), and chitosan (Sigma Aldrich, medium molecular weight) were used for the synthesis of pectin aerogels and pectin aerogels coated with a layer of chitosan. Ethanol absolute, C₂H₅OH (Merck, Darmstadt, Germany) and carbon dioxide, CO₂ ( purity 99.5%, Messer, Ruše, Slovenia) were introduced for the solvent exchange during synthesis of gels and supercritical drying of prepared gels, respectively. Curcumin (purity ≥ 65%, Merck, Darmstadt, Germany) was used as an active substance for prepared aerogels. chitosan was dissolved in acetic acid, CH₃COOH (purity ≥ 98%, Fisher Scientific, Pittsburgh, PA, USA). Hydrochloric acid, HCl (purity 37%, Merck, Darmstadt, Germany), potassium phosphate monobasic, KH₂PO₄ (purity ≥ 98%, Merck, Darmstadt, Germany) and sodium hydroxide, NaOH (purity ≥ 98%, Merck, Darmstadt, Germany) were employed for the preparation of simulated gastric (SGF) and simulated intestinal fluid (SIF) for swelling and drug release studies. SGF (HCl) with pH = 1.2 was prepared by diluting 8.3 mL of 37% HCl to 1000 mL with miliQ water. SIF (phosphate buffer solution) with pH = 6.8 was prepared by mixing 250 mL of 0.2 M KH₂PO₄ and 112 mL of 0.2 M NaOH and diluted to 1000 mL with miliQ water.