Purification and preparation techniques for single

2.2.7.1 Histone preparation

The following protocol was used to isolate single core histone types from HL-60 cells, purify them via size exclusion chromatography (P60-column) and high performance liquid chromatography (HPLC), and to subsequently analyse them by using capillary zone electrophoresis.

For this purpose, 5-6×10⁸ control HL-60 cells and apoptosis induced HL-60 (induction with 150 ng/ml topotecan^® for 8 h) cells were harvested, washed with PBS, and centrifuged at 1,200 rpm for 5 min. Aliquots of about 3×10⁷ cells were taken to isolate the nuclei from which the histones were to be prepared later on. For the nucleus-cytosol preparation, the third protocol (III) by Bunce and co-workers (1988) was used (c.f. above). All nuclei-containing aliquots were then pooled and suspended in 8 ml phosphate-EDTA buffer (5 mM sodium phosphate, pH 6.8, 0.2 mM EDTA, 1 mM PMSF). To make sure that all histones become

detached from the genomic DNA, 1.92 ml of a 5 M NaCl solution were added to the lysate to receive a final salt concentration of 1 M NaCl. The sample was shortly vortexed and kept on ice for 10-15 min. After that, the samples were sonified twice for 15 s at 25% amplitude, to get rid of all genomic DNA, and centrifuged in an SS34 rotor (Sorvall centrifuge) at 15,000 rpm for 25 min at 4°C. The supernatant was removed and dialysed overnight at 4°C in 30 mM HCl, with frequent changes of the buffer to the salt. A membrane with a molecular cut-off of 5,000 kDa was used, to ensure that no histones were lost during dialysis. Subsequently, precipitated unsoluble non-histone proteins were removed from the sample by centrifugation at 13,000 rpm for 10 min at 4°C. The supernatant was carefully removed and the pellet discarded. The clear histone lysate was immediately used for further purification and separation on a P60-column via size exclusion chromatography or stored on ice for a short time.

2.2.7.2 Preparation of a Bio-Gel^® P60 polyacrylamide gel column

Size exclusion chromatography or gel filtration separates soluble proteins according to their size and is based on the molecules’ different permeation properties into porous carrier matrices of defined pore size. Proteins larger than the pore size cannot enter the matrix.

They are eluted together with the void volume. Smaller molecules migrate into the pores of the matrix and are therefore retarded in their elution process.

Bio-Gel P gels are porous polyacrylamide beads prepared by copolymerisation of acrylamide and N,N’-methylene-bis-acrylamide. The matrices are extremely hydrophilic, essentially free of charge and provide efficient, gentle gel filtration of sensitive compounds. A high resolution of protein samples is assured as these columns allow a consistent narrow distribution of bead diameters and excellent molecular weight discrimination.

For the separation of core histones a Bio-Gel P-60 (medium) matrix, which separates proteins with sizes below 60 kDa, was used with the following properties:

particle size range of hydrated beads (µM): 90-180 hydrated bed volume (ml/g) of dry gel: 11

flow rate (cm/h): 4.0-6.0 fractionation range/

nominal exclusion limit (Da): 3,000-60,000

column size: ∅ 1.5 cm; cross length 50 cm; max. sectional area 1.77 cm²; volume 89 ml

The Bio-Gel^® P-60 was prepared by soaking the material in 30 mM HCl overnight. After that the beads were washed three times using large volumes of 30 mM HCl. Once the hydration process was completed, excess volumes of the solution were decanted and the material was

transferred to a filter flask. The flask was connected to a vacuum source to degas the material for 30 min, occasionally swirling it. This was done to avoid trapping air bubbles in the matrix that might have led to an uneven packing of the column. Finally, the slurry was poured into the column in one single smooth movement, to avoid splashing and ensure even packing. Once a 2-5 cm bed had formed, the column outlet was opened to allow packing.

Before actual use 2 bed volumes of buffer were passed through the column.

2.2.7.3 Separation and crude purification of core histones on Bio-Gel^® P60 columns The column flow was adjusted to 0.2-0.3 ml/min. 4 ml of histone extract were applied to the column. Once the sample had entered the gel bed completely, a further volume of 30 mM HCl was added and the elution was started. The actual fractionation process did only start after about 5-7.5 ml of void volume had passed through the column. After that 75 fractions with 1 ml per fraction were collected, using an automatic fraction collector (Amersham Pharmacia). For further analysis 40 µl of each fraction were mixed with 14 µl Laemmli sample buffer and boiled for 5 min. All fractions were subsequently analysed on SDS-PAGE.

After fractionation the column was rinsed with two column volumes 30 mM HCl, sealed tightly with parafilm, to avoid dehydration and stored at room temperature until further use.

The column fractions were pooled in three main fractions with a) H1 linker histones and histone H3, b) histones H2A/H2B and traces of histone H3 and c) mainly histone H4. To reduce the single fraction volumes for further analysis on high pressure liquid chromatography (HPLC), all three fractions were concentrated using Viva Spin concentrators with 20 ml and 6 ml volumes (Viva Science Sartorius group). These have a cut-off of 5,000 Da, to ensure that no protein is lost during the process. All three fractions were concentrated from 20 ml/15 ml to 1 ml per fraction and further purified on an HPLC system.

2.2.7.4 Separation and purification of single core histone types on high performance liquid chromatography (HPLC)

To purify the histones for further single-histone type analysis via capillary zone electrophoresis (CZE), the samples were put through an HPLC system first.

The HPLC separation method for core histones was modified after a protocol originally used by Talasz et al. (Talasz et al., 1998) for the separation of H1 linker histone subtypes. The HPLC equipment used was a Kontron Instruments 450 MT2 system, including an M420 pump, an M425 gradient former, and an M 432 variable wavelength detector. For appropriate cooling of the pump, a perfusor (Perfusor secura, B. Braun, Melsungen) operating with isopropanol was used.

Before loading a 150 µl sample (approx. 100 µg) onto the column, the samples were centrifuged in a table ultracentrifuge (Optima™ TLX 120 CE ultracentrifuge, Beckman Coulter) at 100,000×g for 15 min at 4°C, using a TLA 100 rotor (Beckman Coulter) with carbonated tubes (7×20 mm; 0.2 ml volume). This step was necessary to remove any traces of insoluble material, which might disturb the separation. The histones, dissolved in 30 mM HCl, were separated on a Jupiter C4 column (150×4.6 mm inner diameter; 5 µm particle size, 300 Å pore size; Phenomenex, Aschaffenburg). The samples were eluted at a constant flow rate of 1 ml/min and the absorbance of the effluent was monitored at λ 210 nm. The following solvents were used to form the gradient: solvent A with 0.1% (v/v) trifluoroacetic acid (TFA), solvent B with 59.5% (v/v) acetonitrile in water (HPLC grade) and 0.1% (v/v) TFA. The column was generally rinsed with solvent C, 100% (v/v) methanol (HPLC grade). For the actual separation of the core histones the following multi-step gradient program was used:

time [min] function

rinsing of column solvent C 100%

0 – 5.20 solvent A 100% / solvent B 0%

5.20 – 10.20 solvent A 50% / solvent B 50%

10.20 – 76.20 solvent A 0% / solvent B 90%

The peaks were recorded using the software program Geminyx version 1.91 (Flowspek AG).

The manually collected samples were vacuum-dried overnight in a Speed Vac centrifuge (Speed Vac SC 100, Savant) and stored at -20°C.

2.2.7.5 Analysis of core histones via capillary zone electrophoresis (CZE)

Capillary electrophoresis (CE) encompasses quite a range of different related separation- techniques that use narrow-bore fused-silica capillaries to separate complex arrays of differently sized molecules, proteins among others. High electric field strengths are used to separate proteins on the basis of differences in their charge, size and hydrophobicity. The samples are loaded onto the system by immersing one end of the capillary into a sample vial and applying high voltage. The proteins are detected by an on-column detector at a wavelength of 200 nm.

One of these highly specified separation-techniques is the capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE). Its great advantages compared with conventional electrophoresis techniques lie in the fact that only very small sample volumes and solvent volumes are needed. Besides the method allows short separation times and high resolutions. The separation mechanism is based on differences in the charge-to-mass ratio

of the samples. Homogeneity of the buffer solution and constant field strength throughout the length of the capillary are essential for CZE. The separation itself principally relies on the pH- driven dissociation of acidic groups on the solute or alternatively on the protonation of basic functions on the solute, depending on the properties of the protein analysed.

A P/ACE MDQ capillary electrophoresis system (Beckman Coulter) was used for the analysis of core histones. The conditions were modified and optimised after a method developed by Lindner et al. (Lindner et al., 1995). A fused-silica capillary was used for the separation. The following conditions were applied for separating core histones:

property condition

cartridge temperature 30°C

voltage 10 kV

current approx. 39 µA

separation time 120 min

sample injection by pressure

injection time 2 s

protein concentration of the sample approx. 0.5 mg/ml

absorbance detection λ 200 nm

length of capillary 1.1 m

separation buffer 30 mM H3PO4, 60 mM HClO4, 0.02% (w/v) hydroxypropylmethylcellulose (HPMC), pH 2.0, adjusted with triethylamine

rinse buffers 1N H2SO4; 1N NaOH; 0.1N NaOH; H2O bidest. (HPLC grade)

Table 2.2.7.5 CZE system properties and separation conditions for the analysis of core histones.

The vacuum-dried samples separated by HPLC were dissolved in 30 mM HCl for 15 min.

During that time they were kept on ice to prevent protein degradation. In a final centrifugation step at 13,000 rpm for 10 min at 4°C (microcentrifuge) all traces of insoluble material were removed which might otherwise block up the capillary. Subsequently, the samples were injected into the system. The absorbance data was recorded and analysed by a PC-based integration system (A P/ACE MDQ), operating with Karat 32™ (version 7.0) software.