The enzyme is usually limited to use as a therpeutics because of its own instability and antigenicity. Adenosine deaminase(ADA), which was selected as a model of enzymes in this study, was modified by a pendant-type polyethylene glycol(PEG) in
order to
reduce the nzyme's own disadvantages, and the authors studied about the biochemical characteristics of the pendant type PEG-ADA conjugate to elucidate whether this conjugate could be a more improved and effective therapeutic enzyme having
stability
and
non-antigenicity.
Calf adenosine deaminase was modified with pendant-type polyethylene glycol using cyanuric chloride as a coupler. The covalent attachmant of PEG to ADA altered the kinetic profile of the enzyme in whch the value of Km for adenosine was decreased.
The Km
value of the ADA and PEG-ADA10 were 30μM, 25μM, 25μM against adenosine, respectively. But the PEG-ADA20 exhibited reasonable retention of catvlytic activity with an increased Km, which was 50 μM against adenosine. The ADA and PEG-ADA showed
optimum
in acidic direction.
The ADA and PEG-ADA had same temperture optima of 50℃. Thermal inactivation at the temperature above optima was essentially the same between the native and modified enzymes. PEG appeared to have no effect of stabilizing enzymes on high
temperature.
This suggested that PEG attachment caused some destabilization of the adenosine deaminase structure. PEG-ADA also demonstrated a resistance to the proteolytic digestion by trypsin and chymotrypsin. The electrophoretic mobility in polyacrylamide
gels of
PEG-ADA was reduced as compared to the native enzyme. The unmodified enzymes showed single band, whereas the modified ones formed a mumber of bands throughout the gels. The decreased electrophoretic mobility of PEG-ADA might be due to a
charge-shielding
effect of the hydrophilic PEG shell surrounding the enzyme. In addition, the increased size of the adducts might serve to retard migration in the polyacrylmide gels. The alteration of the pH dependance and the dramatically decreased mobility in
polyacrylamide gel electrophoresis of the PEG-enzyme pointed up the effect of PEG modification on the apparent charge of the enzyme molecules. It was believed to be due to a change in conformation dictated by the charge of the PEG-enzyme or
imposed
by
the covalent attachment of PEG to the enzyme. These results showed that the bochemical properties of adenosine deaminase were altered after chemical modification by pendant type PEG. The preparation of PEG-ADA retained the enzyme activity and had
resistance against proteolytic enzymes.
The authors concluded that the pendent-type PEG-ADA might be available in the medical field in the future.
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