3. Material and Methods
3.2. Reagents and Buffers
The following Tables 3.6 – 3.7 shows reagents used for this work. Unless otherwise stated, reagents were purchased in p.a. quality.
Table 3.6: Chemicals and reagent used for the investigation of Allium species.
Reagent Source
1,1-Diphenyl-2-picrylhydrazine Sigma (Sigma-Aldrich Chemie), Steinheim
2-Methyl-2-propanthiol Merck, Darmstadt
Acetic acid Merck, Darmstadt
Acetonitrile HPLC gradient grade SDS Peypin/France
Boric acid Merck, Darmstadt
Bovine serum albumin Merck, Darmstadt
Reagent Source
Bradford reagent Sigma (Sigma-Aldrich Chemie), Steinheim
Butanol Merck, Darmstadt
Butylated hydroxytoluene Sigma (Sigma-Aldrich Chemie), Steinheim Calcium chloride dihydrate Merck, Darmstadt
D-(+)-Saccharose Sigma (Sigma-Aldrich Chemie), Steinheim D-(+)-Glucose monohydrate Merck, Darmstadt
Dichloromethane Merck, Darmstadt
di-Sodium hydrogen phosphate dihydrate Merck, Darmstadt di-Sodium hydrogen phosphate dihydrate
Suprapur®
Sodium hydroxide Suprapur®
Merck, Darmstadt Merck, Darmstadt di-Sodium hydrogen phosphate
monohydrate
Merck, Darmstadt
Ethanol Merck, Darmstadt
Ethyl acetate Merck, Darmstadt
Folin-Ciocalteu´s phenol reagent Fluka (Sigma-Aldrich Chemie), Steinheim
Chloroform Merck, Darmstadt
Magnesium chloride hexhydrate Merck, Darmstadt Manganese chloride dihydrate Merck, Darmstadt
Methanol SDS Peypin/France
n-Hexane Merck, Darmstadt
Ninhydrin Merck, Darmstadt
o-Phtaldialdehyde for fluorimetry Fluka (Sigma-Aldrich Chemie), Steinheim o-Phtaldialdehyde for synthesis Merck, Darmstadt
Potassium di-hydrogen phosphate Merck, Darmstadt
Reagent Source
Potassium-sodium-tartrate Merck, Darmstadt
Sea sand Fluka (Sigma-Aldrich Chemie), Steinheim
Sodium azide Merck, Darmstadt
Sodium carbonate Merck, Darmstadt
Sodium dihydrogen phosphate dihydrate Merck, Darmstadt Sodium dihydrogen phosphate dihydrate
Suprapur ®
Merck, Darmstadt
Sodium hydroxide Merck, Darmstadt
Sodium chloride Merck, Darmstadt
Sodium chloride Suprapur® Merck, Darmstadt Sodium tetraborate decahydrate Merck, Darmstadt
Thioglycolic acid Fluka (Sigma-Aldrich Chemie), Steinheim
Thiomersal Fluka (Sigma-Aldrich Chemie), Steinheim
Concanavalin A Sigma (Sigma-Aldrich Chemie), Steinheim
NADH Merck, Darmstad
Pethrolether (40-60 °C) Riedel de Haen
Pyridoxal-5´-phosphate Merck, Darmstadt
LDH suspension (18.5U/5 µl) Sigma (Sigma-Aldrich Chemie), Steinheim
Silica gel 60 Merck, Darmstadt
Ammonium sulphate, 99,99% Merck, Darmstadt
Table 3.7: Cysteine sulphoxides used as reference compound in this investigation (HPLC and FIA).
Substance (+) Enantiomer [%] (-) Enantiomer [%] Lot
Methiin 38.9 61.1
Ethiin 47.5 52.5 Alliin >97
>97 50
<3
<3 50
MK-02-108-01
MK-02-156-01
Propiin 100 61.5
0 38.5
Buthiin 49.1 50.9
Hexiin 40.5 50.5
For biosensoric FIA experiments, water for analysis (water p.a.) purchased from Merck, Darmstadt was used. For other experiments Millipore® water was used.
In the following Tables 3.8-3.9, buffers used for chromatographic separations, biosensoric FIA experiments and radical scavenger acitivity testing are listed.
Table 3.8: Buffers and solutions used for chromatographic purposes.
Name Reagents Quantity Method of preparation Borate-buffer
pH 9.5
Sodium tetraborate decahydrate
4.77 g The reagent was dissolved in 200 ml of Millipore water and pH was regulated with a solution of sodium hydroxide. The volume was adjusted to give 250.0 ml.
Phosphate-buffer pH 6.5
Sodium di-hydrogen phosphate dihydrate di-Sodium hydrogen phosphate monohydrate
1.93 g
2.53g
Both reagents were dissolved in 1000.0 ml of Millipore water. If needed, pH was regulated.
Solution for derivatization
o-Phtaldialdehyde for synthesis
Methanol
2-Methyl-2-propanthiole
140 mg
5 ml 200 µl
o-Phtaldialdehyde was dissolved in methanol.
2-Methyl-2-propanthiole was added and then Borate buffer (in small portions). The work had to be performed in a fume hood.
Name Reagents Quantity Method of preparation Borate buffer pH 9.5 50 ml
Solution of sodium
hydroxide (1M)
Sodium hydroxide 4.00 g Reagent was dissolved in 100.0 ml of Millipore water
Solution of ninhydrin
Ninhydrin Butanol
Acetic acid 98%
30 mg 10 ml 0.3 ml
After the complete dissolution of ninhydrin in butanol acetic acid was added.
Table 3.9: Buffers and solutions used for biosensoric FIA experiments and determination of radical scavenger activity.
Name Reagents Quantity Method of preparation Stock solution
of allin 0.001 M
Alliin (99%) 18.8 mg Alliin was dissolved in 100.0 ml of biosensoric eluent.
Stock solution of ammonium sulphate 0.001 M
Ammonium sulphate 99.99%
13.2 mg Reagent was dissolved in 100.0 ml of biosensoric eluent
Biosensor buffer
Sodium dihydrogen phosphate dihydrate Suprapur ®
di-Sodium hydrogen phosphate dihydrate Suprapur®
Sodium chloride Suprapur®
Thiomersal
888 mg
1.776 g
10 g
100 mg
Reagents were dissolved in 800 ml of water p.a. The volume was adjusted to give 1000.0 ml.
Con A-buffer Phosphate-buffer pH 7 (600 mM)
Sodium chloride
Calcium chloride dihydrate Magnesium chloride hexyhydrate
Manganese chloride dihydrate
Pyridoxal-5´-phosphate Thiomersal
100 ml
58.4 g 147.0 mg 203.3 mg
161.8 mg
26.5 mg 200 mg
Phosphate buffer and the salts were dissolved in 800 ml of water p.a. After 10 minutes Pyridoxal-5´-phosphate and thiomersal were added.
Solution was filtered. The volume was adjusted to give 1000 ml.
OPA buffer pH 9.0
o-Phtaldialdehyde for fluorimetry (OPA)
1.006 g OPA was suspended in 200 ml of water p.a. Thioglycolic acid
Name Reagents Quantity Method of preparation (15 mM)
Thioglycolic acid 2.73 ml
was added. After dissolution of the OPA, the pH was regulated with solution of sodium
hydroxide for FIA purposes.
The volume was adjusted to give 500 ml. The work had to be performed in a fume hood.
Boric acid buffer (0.8 M) pH 12
Boric acid 24.732 g Boric acid was suspended in 400 ml of water p.a. The pH was regulated with a solution of sodium hydroxide for FIA purposes. The volume was adjusted to give 500 ml.
Solution of sodium hydroxide for FIA purposes (1M)
Sodium hydroxide Suprapur®
4.0 g Sodium hydroxide was dissolved in 100 ml of water p.a.
Phosphate buffer pH 7 (600 mM)
Potassium di-hydrogen phosphate
di-Sodium hydrogen phosphate dihydrate
35.4 g
72.52 g
Reagents were dissolved in 800 ml of water p.a. The volume was adjusted to give 1000.0 ml.
Whenever necessary, pH was regulated.
Sørensen buffer (60 mM)
Phosphate buffer pH 7 (600 mM)
Thiomersal
100 ml
0.2 g
Thiomersal was dissolved in 200 ml of water p.a. and the phosphate buffer pH 7 was added. The volume was adjusted to give 1000.0 ml.
Alliinase Buffer Phosphate buffer pH 7 (600 mM)
Saccarose Sodium chloride
Pyridoxal-5´-phosphate Thiomersal
100 ml
100 g 10 g 26.5 mg 0.2 g
Phosphate buffer pH 7 and the reagents were dissolved in 800 ml of water p.a. After 10
minutes,
pyridoxal-5´-phosphate and thiomersal were added. The volume was
adjusted to give 1000.0 ml.
NADH solution (8.5mM)
NADH
Sørensen buffer
12.0 mg 2.0 ml
NADH was dissolved in Sørensen buffer.
LDH solution LDH suspension Sørensen buffer
150 µl 2.0 ml
LDH was dissolved in Sørensen buffer.
DPPH solution 1,1-Diphenyl-2-picrylhydrazine
0.01 g 1,1-Diphenyl-2-picrylhydrazine was dissolved in 25 ml of methanol p.a. The dissolution took 2 hours. A dark graduated flask had to be used.
Name Reagents Quantity Method of preparation BHT solution Butylated hydroxytoluene 0.08 g Butylated hydroxytoluene was
dissolved in a small amount of methanol p.a. The volume was adjusted to give 25.0 ml.