ANALYTICAL WOOCEMOTHY 162, 000-000 ( 1 987) 
Separation of Human from Mouse and Monkey Adenosine 
Deaminase by lon-Excbange Chromatography Following 
Retrovirai-Mediated Gene Transfer 
Jeanne R. McLachlin, Sheri C. Bernstein, and W. French Anderson 
Laboratory of Molecular Hematology. national Heart. Lung, and Blood Institute. 
Hational Institutes of Health. Bethesda. Maryland 20892 
Received September IS, 1986 
A method for the chromatographic leparadoa of human adenosine deaminase (ADA) from 
murine and monkey ADA is described. This procedure was developed in order to detect the 
expression of low or moderate levels of human ADA following retro viral- media ted gene transfer 
of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation 
was achieved oo a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein 
liquid chromatography system and was found to be a highly reproducible method yielding 
enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M 
potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human 
ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KG), while murine 
ADA elutes at 0.12 m KG and monkey ADA at 0.15 M KC1. The column fractions were assayed 
for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and 
monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and 
reproducible separation of human ADA from that of other species and yields partially purified 
enzymatically active protein, e imt Aerntmc Fnm. lac 
Key Words; adenosine deaminase; ion-exchange chromatography, FPLC. TLC 
Adenosine deaminase (ADA 1 ; EC 3. 5. 4. 4) 
is an important enzyme of purine metabo- 
lism, catalyzing the deamination of adeno- 
sine and deoxyadenosine (1). In man, an ab- 
sence of ADA activity is associated with one 
form of severe combined immunodeficiency 
diseas e (ADA - SCID; reviewed in (2,3)). The 
ADA protein has been identified and charac- 
terized from a variety of sources including 
amphibians (4,5), birds (6), and mammals 
(7-12). In humans, ADA is a ubiquitous 
1 Abbreviations used; ADA, adenosine deaminase; 
DCF, 2'-deoxycoformycin; EHNA, erythro-9-{2-by- 
droxy-3-nonyl)adeninc; MTT, (3-(4,5-dimethylthiazol- 
2-y1}-2,5-dipbenyltetrazolium bromide: Thiazolyl blue); 
RBC, red blood cell; PBS, phosphate-buffered saline; 
FPLC, fast protein liquid chromatography, LSM, Lym- 
phocyte Separation Medium; Buffer A. 0.05 M potas- 
sium chloride. 20 mM Tris-HG, pH 7.5; Buffer B, 1.0 M 
KQ. 20 mM Tria-HO, pH 7.5. 
protein with several isozymes that differ in 
their electrophoretic mobilities (13-16). 
Human red blood cell (RBC) ADA presents 
a characteristic triple-banded pattern when 
analyzed by starch gel electrophoresis (9), 
while ADA from other tissues is composed of 
isozymes which do not migrate toward the 
anode as far as RBC ADA (13). Human 
erythrocyte ADA also exhibits multiple elec- 
trophoretic forms by isoelectric focusing (15) 
corresponding to a single polypeptide chain 
with an approximate molecular weight (A/ r ) 
of 38,000 (17) which has undergone variable 
degrees of glycoslyation (16). A larger form 
of human ADA (A/ r 298,000) has been iso- 
lated from several tissues (15,18) and deter- 
mined to be a complex of the 38,000 M, 
ADA species and a larger binding protein 
(M, 200,000). The large form is predominant 
in tissues which exhibit lower ADA specific 
0005-2697/87 J3.00 
Copyright O I»I7 by Academic Ni. lac 
AJ ngha at nproducooa ia ay bra raaarvad. 
Recombinant DNA Research, Volume 12 
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