Chromatographic and electrophoretic methods for protein separation and

3.4.1 Reverse Phase High Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) was used in the present work to separate components of a mixture by using different chemical interactions between the protein/peptides of interest and the chromatography column. Molecules bearing some extent of hydrophobic character, such as proteins and peptides, can be separated by reverse phase chromatography with good resolution and recovery. RP-HPLC is based on the principle of hydrophobic interactions, resulting from repulsive forces between a polar eluent and the non-polar stationary phase.

Usually, the sample is injected into the HPLC column directly into the stream of mobile phase and it is retarded by specific chemical or physical interactions with the stationary phase as it pass through the length of the column. There are mainly three types of columns namely, C4, C8, and C18 that interact with the hydrophobic moieties of the analyte. The C4 and C8 are used for protein separation, while C18 is used for small peptides. Peptides and/or proteins are eluted from the reverse phase column with aqueous solvents containing an ionic modifier to adjust the pH and an organic modifier to displace and elute the peptide by increasing gradient in the organic modifier as shown in Table 3.12.

Table 3.12. The gradient used for the separation of synthetic peptides Time %A %B

0 100 0

5 100 0

85 20 80

90 0 100

95 100 0

100 100 0

Prior using aqueous solutions, they were well degased by vacuum combined with sonication. The sample was dissolved in 0.1% TFA compatible with the mobile phase (usually the components of the gradient starting point) to avoid precipitation in the pores of the column packing. All RP-HPLC experiments were performed on a Bio-Rad system (Bio-Rad Laboratories, Richmond, CA) using PLRP-S column (250 x 4.6mm, 300 Å, 5µm) contain rigid macroporous spherical particles of polystyrene/divinylbenzene (Polymer Laboratories, Darmstadt, Germany). The

samples were dissolved in eluent A and the peaks were detected at two different wavelengths λ = 365 nm for nitrated peptides and λ = 220 nm for non-nitrated peptides. The purification of polypeptides was carried out on a Knauer system (Bad Homburg, Germany) using a preparative C18 column (GROM-SIL 120 ODS-4 HE, 10 µm, 250 x 20 mm, pore size 120 Å; Herrenberg-Kayh, Germany) The same eluents as described above with appropriate linear gradients were applied. Flow rate was 10 mL/min for preparative HPLC. Peaks were detected at 365 nm and 220 nm.

3.4.2 Sodium dodecyl sulphate – polyacrylamide gel

A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS (also called lauryl sulfate) is an anionic detergent, meaning that, when dissolved, its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecuar mass. The negative charges on SDS destroy most of the complex structure of proteins, and they are strongly attracted toward an anode (positively-charged electrode) in an electric field.

SDS PAGE was performed accoding to Laemmli using the Mini-Protean II gel electrophoresis system (Biorad, München, Germany) by pouring SDS-polyacrylamide gels between two glasses plates resulting a gel were 90 x 60 x 1 mm in size. The stacking gel was poured over the top of the separating gel, which allows the proteins in a lane to be concentrated into a tight band before entering the separating gel. The acrylamide concentration stacking gel is lower than the concentration of separation gel and thus a larger pore size, lower pH and different ionic content. The lower the concentration of acrylamide the larger size of the pores in the gel is created, therefore gels with a low percentage of acrylamide are typically used to separate large proteins, while high percentage gels were used for small proteins. Table 3.13 shows the different acrylamide concentrations used in the present work.

Table 3.13. Composition of SDS-polyacrylamide gels according to Laemmli.

Stacking gel Separating gel Monomer concentration

c 30% (w/v) Acrylamide, 0.8% (w/v) N, N’- Methylenebisacrylamide

d 10% (w/v) Ammonium persulfate

e N, N, N’, N’- tetramethylethylenediamine

Muscle proteins were solubilised in a SDS reducing buffer (50 mM Tris-HCl, 4 % (w/v) SDS, 25 % (w/v) glycerol, 6M urea, 0.02 % (w/v) bromophenol blue, pH 6.8). The running buffer used contained of 25 mM Tris, 192 mM Glycin and 0.1 % SDS. Gel electrophoresis was carried out using a Power/PAC 1000 power supply (Bio-Rad, München, Germany) at a constant voltage of 60 V for approximately 30 min, until the tracking dye entered the separating gel, and at 110 V for ca. 2 hrs., until the tracking dye reached the anodic end of the separating gel. After separation, proteins were visualised by sensitive colloidal Coomassie or transferred on PVDF membrane for Western blot experiments. The Coomassie gels were scanned using a GS-800 Calibrated Imaging Densitometer (Bio-Rad, München, Germany). The molecular weights of proteins of interest were estimated by running standard proteins of known molecular weights, summarized in Table 3.14.

Table 3.14. Protein molecular weight marker for SDS-PAGE analysis (PS-101, Jena Bioscience) Molecular weight marker proteins Molecular weight (Da)

Phosphorylase b 97,400

Bovine serum albumin 66,200 Alcohol dehydrogenase 37,600

Carbonic anhydrase 28,500

Myoglobin 18,400 Lysozyme 14,400

3.4.3 Two- dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-D gel electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. By this method proteins are separated in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (Mw). In this way, complex mixtures consisting of thousands of different proteins can be resolved and the relative amount of each protein can be determined.

The procedure involves placing the sample in gel with a pH gradient, and applying a potential difference across it. In the electrical field, the protein migrates along the pH gradient, until it carries no overall charge. This location of the protein in the gel constitutes the apparent pI of the protein. The IEF is the most critical step of the 2-D electrophoresis process. The proteins must be solubilized without charged detergents, usually in high concentrated urea solution, reducing agents and chaotrophs. To obtain high quality data, it is essential to achieve low ionic strength conditions, before the IEF itself. Since different types of samples differ in their ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of sample. The main experimental steps involved in 2-D gel electrophoresis are shown in Figure 3.61.

Figure 3.61. Two-dimensional gel electrophoresis experimental steps. Crude protein mixture extracts are separated in the first dimension according to their specific isoelectric point (pI), followed by the separation in the second dimension by their molecular weight (Mw).

IEF was carried out using a Multiphor horizontal electrophoresis system (Amersham Biosciences, Uppsala, Sweden) using 17 cm IPG strips (pH range 3-10) with the sample being applied overnight using the in-gel rehydration method. The reswelling solution contained 7 M urea, 2 M thiourea, 4 % CHAPS, 0.3 % DTT, 2 % Servalyt (3-10) and a trace of bromophenol blue. Rehydrated strips were run in the first dimension for about 30 kVh at 19 ^oC. After focusing, the IPG strips were equilibrated for 30 min in 6 M urea, 30 % glycerol, 2 % (w/v) SDS, 0.05 M Tris-HCl (pH 8.8), 1 % (w/v) DTT and a trace of bromophenol blue, and they were then run for further 30 min in the same solution except that DTT was replaced by 4.5 % (w/v) iodoacetamide. The second dimensional separation was carried out with a Bio-Rad Protean II xi vertical electrophoresis system using 12 % SDS-PAGE gels of 1.5 mm thickness. Strips placed on the vertical gels were overlaid with 1 % (w/v) agarose in SDS running buffer (25 mM Tris-HCl, 192 mM glycine and 0.1 % (w/v) SDS), and subjected to electrophoresis at 25 mA /gel for 30 min and 40 mA/gel until the tracking dye reached the anodic end of the gel. After separation in SDS-PAGE gels, the proteins were visualized by sensitive Colloidal Coomassie staining and scanned using a GS-800 calibrated imaging densitometer (Bio-Rad, München, Germany).

3.4.4 Colloidal Coomassie staining

Coomassie Blue staining is based on the binding of the dye Coomassie Brilliant Blue G250 (Figure 3.62), which binds non-specifically to virtually all proteins.

Although Coomassie Blue staining is less sensitive than silver staining, it is widely used due to its convenience. The gel is soaked in a solution of the dye. Any dye that is not bound to protein diffuses out of the gel during the destaining steps. Coomassie blue binds to proteins approximately stoichimetrically, so this staining method is preferable, when relative amounts of protein need to be determined by densitometry.

The dye molecules are forming complexes with basic amino acids such as arginine, tyrosine, lysine and histidine making the proteins detected as blue bands on a clear background.

Figure 3.62 Coomassie Brilliant blue G250

Below is described the Coomassie staining procedure representing the modified method introduced by Neuhoff [16] :

1. Fix the gel with gentle agitation for at least 1 h in 12 % (w/v) TCA.

2. Prepare the colloidal Coomassie stain by mixing 200 ml solution of 10%

(w/v) ammonium sulphate and 2 % (w/v) phosphoric acid in MilliQ water with 2 ml of 5 % Coomassie Brilliant Blue G250 dissolved in MilliQ water (ammonium sulphate increases the strength of hydrophobic interactions between proteins and dye; the methanol allows a much faster staining process). Shake this solution strongly for a few minutes and then add 50 ml methanol. Continue shaking until the first step is ready.

3. Remove the fixation solution and place the gel in the freshly prepared colloidal Coomassie stain. Stain the gel overnight with gentle agitation.

4. Wash the gel with 25 % methanol in MilliQ water for 1 h.

5. Wash the gel with MilliQ water.

6. Repeat step 5 until the protein bands are at the desired contrast against the background of the gel.

3.4.5 Silver staining

Silver staining is the most sensitive staining method for permanent visible staining of proteins in polyacrylamide gels. This sensitivity, however, comes at the expense of high susceptibility to interference from a number of factors. In silver staining, the gel is impregnated with soluble silver ions and developed by treatment with formaldehyde, which reduces the silver ions to form an insoluble brawn precipitate of metallic silver, followed by further autocatalytic reduction and consecutive image formation, which is finally stopped by immersing the gel in a solution of glycine or acetic acid in water. Silver stains can be acidic (with silver nitrate) or basic (with silver diamine). In this work, the silver staining procedure used was according to Heukeshoven and Dernick (Table 3.15) in which aldehydes were omitted in fixative and sensitization steps in order to enable mass spectrometric characterization of proteins.

Table 3.15. The modified silver staining protocol according to Heukeshoven and Dernick.

Step Solution

(100 ml/1D^a gel; 250 ml/2D^b gel) Time (min.) Fixation 30 % (v/v) ethanol, 10 % (v/v) acetic

acid in MilliQ water > 30 Sensitivization 30 % (v/v) ethanol, 6.8 % (w/v)

sodium-acetate, 0.2 % (w/v) Na2S2O3 x 5H2O in MilliQ water

30 min. or overnight

3 X wash Milli Q /water 3 X 15

Silver 0.2 % (w/v) silver nitrate in MilliQ water, 50 µl formaldehyde (dark)

20 Develop 1 100 ml 2.5 % (w/v) Na2CO3 in MilliQ

water, 10 µl formaldehyde (volume for 2D^b)

1 Develop 2 200 ml 2.5 % (w/v) Na2CO3 in MilliQ

water, 20 µl formaldehyde (volume for 2D^b)

3 to 7 Stop 1 % glycin in MilliQ water 10

3 X wash Milli Q / water 3 X 15

a1D: one-dimensional gel electrophoresis.

b2D: two-dimensional gel electrophoresis.

3.4.6 BioAnalyzer Gel reader for native fluorescence detection

After separations (SDS-PAGE and 2-D gel), proteins were visualized using also the Gel-BioAnalyzer instrument. The structure of the Gel-BioAnalyzer (LaVision-Biotec GmbH, Bielefeld, Germany) is schematically shown in Figure 3.63. The principle of Gel-BioAnalyzer is based on a UV excitation source and a detection system within the UV range. The UV excitation light was generated by a 300 W xenon lamp (265 - 680 nm). The irradiation area was set to 1 cm² at 35 mW/cm² and imaged by three lenses onto a photomultiplier detector. A UV bandpass filter (280 - 400 nm) is incorporated to block the excitation light from the detection system. From four filter positions (one for UV excitation, three for visible fluorescence), the UV filter transmitting light at λ = 343 ± 65/2 nm was employed. The large reading area (30 × 35 cm²) provided scanning of both one- and two-dimensional gels. The instrument is equipped with removable gel tray and it is able to read unstained as well as stained protein gels. In the present work only scanning of unstained gels was employed.

After fixation in position on the gel tray, localisation and isolation of gel spots was carried out by moving the gel tray, with positioning and scanning of the gel controlled by the LaVision-Biotec scanning software.

Figure 3.63. Scheme of the BioAnalyzer Gel instrument, adapted after http://www.lavisionbiotec.com/

en/ microscopy-products/gelreader

3.5 Chemical modification reaction and enzymatic fragmentation of proteins