cupressoides was fractionated by DEAE-cellulose
anion-exchange chromatography applying a stepwise of NaCl, yielding three SPs fractions (-SP1, -SP2 and Cc-SP3 eluted at 0.50, 0.75 and 1.00 M NaCl, respectively), based on their respective metachromatic properties (Rodrigues et al., 2011a).
terreus culture (A) and Ion-exchange chromatography on DEAE-cellulose
of the major active L-asparaginase component obtained from gel filtration (B)
Interestingly, only two major peaks, eluted from the DEAE-cellulose
column with 0.5 and 0.75 M of NaCl, respectively, were obtained for Am.E-2.
The method of producing the inhibitor in purified form comprised the following steps: extraction of the enzyme, ultrasonic disintegration, ion exchange chromatography on DEAE-cellulose
52, dialysis, freeze-drying (Ukrainian Patent N 89778) (Divocha, 2010).
concentrated solution containing the G6PD activity was applied onto a Sephacryl S-300 column (142 cm x 1.75 cm i.d.).
Crude extract was further purified by ion exchange chromatography on DEAE-cellulose
column using 20mM Tris buffer, pH 8.
Enzyme purification by DEAE-Cellulose
Essentially, two grams of dehydrated algal tissue cut in small pieces were subjected to papain digestion (60[degrees]C, 24 hours) as previously described (Rodrigues et al., 2010), followed by fractionation of a sample of extract (20 mg) dissolved in 10 mL of 50 mM sodium acetate buffer (pH 5.0) by anion-exchange chromatography (DEAE-cellulose
), where the column (1.2 X 12 cm)-bound SPs were eluted with NaCl (from 0 to 2 M, with 0.25 M of intervals) added to buffer of equilibrium and the collected fractions (2.5 mL) checked for SPs using metachromatic assay containing dimethymethylene blue in an Amersham Bioscience Ultrospec 3100 spectrosphotometer at 525 nm (Farndale, Buttle, & Barrett, 1986).
The crude SP was extracted from the dehydrated algal tissue (room temperature) by papain digestion (60[degrees]C, 6h), and then subjected to fractionation by anion-exchange chromatography on a DEAE-cellulose
column using a NaCl gradient (0 [right arrow] 1 M, with 0.25 M of intervals).
Secondly, the CS was purified by DEAE-Cellulose
52 chromatography and Sephadex G-100 chromatography to afford purified CS.
The purification protocol entailed ion exchange chromatography on DEAE-cellulose
. SP-Sepharose, and Q-Sepharose and gel filtration on Superdex 75.
It would be interesting to see if the Hb [A.sub.2] concentrations in her Hb D-containing samples are again on the low side of normal when analyzed with other procedures (DEAE-cellulose
chromatography or cellulose acetate elution procedure).