Effect of molecular architecture of poly(N-isopropylacrylamide)-trypsin conjugates on their solution and enzymatic properties.
Polymer-enzyme hybrid conjugates modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), have been synthesized. We have investigated the molecular architecture of PIPAAm-enzyme conjugates by preparing two types of PIPAAm-trypsin conjugates, wherein PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to trypsin by single-point conjugation (IDc-trypsin). A copolymer (PIDAAc) consisting of acrylic acid and IPAAm randomly linked in polymer chains was attached to trypsin using multipoint conjugation (PIDAAc-trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free polymer end group which remains sensitive to small temperature changes. The PIDAAc-trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in solution was improved after introduction of PIPAAm chains, which prevented autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-trypsin conjugate was more stable than the PIDAAc-trypsin. As a consequence, single-end conjugation of polymer to enzyme provides novel bioconjugate with novel functionality attributed to attached polymer while retaining native biological function with high stability.
Matsukata
M
M
Department of Chemistry, Sophia University, Tokyo, Japan.
Aoki
T
T
Sanui
K
K
Ogata
N
N
Kikuchi
A
A
Sakurai
Y
Y
Okano
T
T
D000180:Acrylic Resins; D004795:Enzyme Stability; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D012995:Solubility; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014867:Water
D000180:Acrylic Resins; D014867:Water; C052970:poly-N-isopropylacrylamide; D014357:Trypsin