3.2.1 Isolation o f Crude G T F Extract
The isolation procedure began with various starting materials. These were live brewers yeast cells (Lion Breweries, Palmerston North, New Zealand), dried powdered brewers yeast (Dominion Breweries, Mangatainoka, New Zealand) and dried torula yeast granules (Sigma cat YCU). The whole yeast cells were harvested and washed as for the assay yeast (Section 2.2. 1 ) before the initial extraction while the other yeasts were used directly in the extraction.
One kilogram of yeast was suspended in 4 litres of 50% ethanol and this was then heated just to boiling and allowed to cool overnight. The yeast was filtered off and the ethanol was removed from the eluate using a rotary evaporator . The remaining aqueous solution was adjusted to pH 3.5 with HCI and treated with 250 g of activated charcoaL After one h our the charcoal was filtered off and the cake was washed with doubly distilled deionised water and 95% ethanol and the filtrates were discarded. The crude GTF was then eluted from the charcoal using 2 litres of diethyl ether: concentrated NH40H. ( 1 : 1 ) for three hours after which the charcoal was again filtered off and the diethyl ether and the
NH40H were removed from the filtrate using a rotary evaporator. The separation up to the stage was based on the procedure of Toepfer et al. ( 1 977) and the material obtained is referred to in further work as the crude GTF extracts.
3.2.2 Paper Electrophoresis
To further purify th� crude G TF extract it was decided to use preparative high voltage paper electrophoresis. It has been claimed that GTF is a cationic compound (Mertz 1 969, Toepfer 1 977) and so separation on the basis of charge is important. It was decided to run the electrophoresis in pH 6.5 buffer to achieve separation of the cationic, amphoteric and anionic components of the crude GTF extract. This technique also had the advantage of separating the crude GTF into a reasonably large n u mber of fract i ons and it had the
capacity to process relatively large amounts of extract so that sufficient material would be available for further purification.
In this procedure up to 200 mg of the crude GTF extract was dissolved in doubly distilled deionized water and loaded in a band along an origin line which evenly divided a sheet of Whatman 3 MM chromatography paper in two. A margin of 50 mm was left on the line on each edge of this band. In this space on each side were loaded two individual standard chromatography marker spots. One of these contained the amino acids asp,
glu, thr, ser, ile, ala, his and gly and the other contained tyr, phe, met, leu, val, arg and lys. Following this, spots of a fluorescent marker containing dansyl-arg and dansyl-OH were placed at 50 mm intervals over the entire length of the line so that the movement of the migrating electrophoretically separated bands could be monitored. The paper was then wetted with the pH 6.5 buffer which was composed of 1 0% pyridine and 0.4% acetic acid v/v. The buffer was applied by pipette in such a way that the liquid migrated by capillary action through the paper and converged from both sides of the sample b and so as to focus the sample material onto the origin as i t became wetted. The wetted electrophoretogram was then lowered into the electrophoresis tank so· that each end was in contact with the reservo:rs of pH 6.5 b uffer and that the centre section of it was in an inert petroleum spirits medium. A potential of 3 kV was then applied across the electrophoretogram\, with the current varying between 200 and 300 mA, for 35 minutes and it was then removed from the tank and allowed to dry. The location of the separated bands was revealed by cutting a strip from each side of the paper which included both markers and an approximately 25 mm wide s trip of the separated sample material. As
GTF was thought to contain pep tides or amino acids it was decided to reveal the separated material on the margin strips using ninhydrin.
3.2.3 Elution o i Fractions From Paper
Material was eluted from the paper b y gravity fed capillary action and the material collected was freeze dried and weighed. As elution of ion exchange columns with NfLtOH h ad shown the ability to activate previously inactive inactive fractions (Haylock 1 983a) and had also shown a direct effect upon the yeast assay (section 2) it was decided to investigate alternative elutants. Other elutants investigated were H20 and 1 % acetic acid.
3.2.4 HPLC o i Electrophoresis Fractions
Electrophoresis fractions showing high levels of activity in the yeast assay were further purified by reverse phase high performance liquid c hromatography (HPLC). The system used consisted of two Waters 6000a pump units controlled by a Waters model 660 solvent programme. The sample was injected using a
U K6
injection system and the column used was a Waters 8MBC 1 8- 10 J.lm (RCM ).Varying amounts of sample were dissolved in water and applied to the column and they were eluted using a linear gradient ( 1 - 100%) starting with 0. 1 M ammonium bicarbonate and changing to 0. 1 M ammonium bicarbonate : isopropanol : aceton itrile ( 1 : 1 : 1 ) with a flow rate of 1 cm3fmin for 40-60 minutes. The eluted material was detected by monitoring absorbance at 220 nm and material was pooled according to the peaks shown by this monitoring.