CHROMATOGRAPHIC SEPARATION OF ADENOSINE DEAMINASE PROTEINS 
the cells were washed in PBS they were 
counted and subsequently lysed by repeated 
freeze-thawing in 200-500 m! 0.05 M potas- 
sium chloride, 20 mM Tris-HQ, pH 7.5. 
Chromatography. A fast protein liquid 
chromatography (FPLC) system from Phar- 
macia (Uppsala, Sweden) was used through- 
out this study. The apparatus consisted of 
two high-pressure P-500 pumps, an 
LCC-500 gradient controller, and an MV-7 
valve sample injector. The absorbance at 280 
nm of the effluent was monitored contin- 
uously with a single-path uv flow cell cou- 
pled to a strip-chart recorder. A Pharmacia 
Mono Q (HR 5/5) anion-exchange column 
was equilibrated with Buffer A (0.05 M potas- 
sium chloride, 20 mM Tris-HC, pH 7.5), 
and the sample (500 m 1 total volume) was 
loaded onto the column at a flow rate of 0.5 
ml/ min. The column was then washed with 
10 ml of Buffer A at the same flow rate fol- 
lowed by a 45-ral increasing linear gradient 
of KQ (0.05-0.5 M) in 20 mM Tris-HCl, pH 
7.5. Finally, the column was washed with 5 
ml of Buffer B (1.0 M KQ, 20 mM Tris-HQ, 
pH 7.5) to remove any remaining proteins. 
All buffers were Altered through a Nalgene 
0.2'tim filter unit (Nalge Co., Rochester, 
NY) and degassed prior to use. Each col- 
lected fraction (1.0 ml) or combined frac- 
tions were then assayed for ADA activity 
using one or more of the methods outlined 
below. 
Starch gel electrophoresis. A procedure for 
the electrophoretic separation of human, 
mouse, and monkey ADA using starch gels 
was adapted from the method of O’Brien et 
al. (29). Gels were prepared using 12% starch 
(Electro-starch Co., Madison, Wl) buffered 
with 1.7 mM citric acid, 6.0 mM Tris-HQ 
(pH 7.1) and were run at 4*C for 18-20 h 
(150 V) in a Buchler vertical gel apparatus 
(Buchler Instruments, Fort Lee, NJ). The 
running buffer was 0.025 M citric acid, 0.085 
M Tris-HQ, pH 7.1. The ADA enzyme was 
detected on the cut surface of the gel using an 
agar overlay method (9). The agar contained 
80 mg of adenosine, 2.0 units of nucleoside 
phosphorylase, and 0.8 unit of xanthine oxi- 
dase (all from Boehringer-Mannheim); 15 
mg of tetrazolium salt; M IT’ and 10 mg of 
phenazine methosulfate (both from Sigma); 
and 1.8 g Difco-Bacto agar (Difco Labs, De- 
troit, MI) in 100 ml of 0.025 M potassium 
phosphate (pH 7.5). 
Thin-layer chromatography. ADA activity 
of individual or combined FPLC column 
fractions was also assayed by measuring 
the conversion of [8- ,4 C]adenosine to 
[8- l4 C]inosine according to the method of 
'Soberman and Karnovsky (30). Column 
fractions were concentrated individually or 
in pairs by ultrafiltration using a Centricon- 
30 microconcentrator (Amicon, Danvers, 
MA). The [8- |4 C]adenosine (sp act 56 mCi/ 
mmol) was lyophilized and resuspended with 
unlabeled adenosine in 100 mM Tris-HQ, 
pH 7.4, to achieve a final concentration of 
1.2 mM adenosine. The reaction mixture 
(consisting of 20 m! of [8- l4 C]adenosine and 
20 pi of concentrate from the FPLC protein 
fraction) was incubated for 2 h at 37°C and 
then heated for 3 min at 100°C to terminate 
the reaction. Samples were centrifuged at 
12,000g in an Eppendorf centrifuge for 5 
min at room temperature and 6 m 1 was ap- 
plied to cellulose thin-layer chromatography 
plates (Kodak No. 13254) on which unla- 
beled carrier adenosine and inosine (10 mM) 
had been applied to the origin. Chromato- 
graphic separation was achieved using 0.1 M 
Na 2 HP0 4 , pH 6.8, saturated ammonium 
sulfate, and n-propyl alcohol (100:60:2) for 
30 min. The adenosine and inosine spots 
were identified with ultraviolet light (254 
nm) and 1-cm 2 spots were cut out, placed in 
vials with 10 ml of Hydrofluor (National 
Diagnostics, Somerville, NJ), and counted in 
a Beckman LS8100 liquid scintillation 
counter. 
RESULTS AND DISCUSSION 
A variety of elution buffers, salt gradients, 
and pH values were tested on an FPLC 
Mono Q column to establish optimal condi- 
Recombinant DNA Research, Volume 12 
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