Open Access Dissertation
Date of Award
Hiroshi Mizukami, Ph.D.
Bovine carbonic anhydrase II (EC 126.96.36.199) was covalently linked to chondroitin-4-sulfate (β-glucoronic acid-[1-3]-N-acetyl-β-galactosamine-4-sulfate-[1-4]) from bovine trachea. A zero length crosslinking procedure which reacts the protein amino groups with preactivated carboxyl groups of chondroitin-4-sulfate was carried out with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide as coupling agents. The complex remained enzymatically active, was water soluble, and appeared to be about 170 kDa as analyzed on SDS-PAGE and column chromatography on Sephadex G 150. It is likely that 5 protein molecules (30 kDa each) are linked to 1 chondroitin-4-sulfate molecule (20 kDa). The CO2 hydration activity of the complex was generally slightly less than the unmodified protein with both forms having highest activity at a pH 8 and the lowest activity at pH 5.5. The same was true for the kinetic parameters kcat and kcat / KM. The pH dependence of kcat was not affected upon conjugation with chondroitin-4-sulfate. The KM appeared to be slightly less for the modified form of the enzyme for the hydration of CO2 at all pH values used. For the dehydration of bicarbonate at pH 5.5 (room temperature), the KM was also slightly less for the modified enzyme (KM = 21.7 mM) as compared to the unmodified form (KM = 24.2 mM). The complex also appeared to be capable of hydrating CO2 in human plasma and seemed to be more resistant of inhibitory effects of plasma as compared to the unmodified enzyme. The effects of a noncompetitive inhibitor, acetazolamide, were similar between the crosslinked complex (KI = 3.87 x 10-8 M) and the unmodified enzyme (KI = 4.62 x 10-8 M). However, the modified enzyme was more resistant to denaturation by guanidine hydrochloride, SDS and heat. Covalent coupling of chondroitin-4-sulfate to enzymes may present a method with which the stabilization of biological structures can be achieved with a minimal (if any) loss of enzymatic activity. The BCA II : chondroitin-4-sulfate complex produced in this work may be of use as an artificial blood substitute designed to transport CO2 molecules in vivo.
Brar, Rajivinder S., "Zero length crosslinking of bovine carbonic anhydrase II and chondroitin-4-sulfate" (1998). Wayne State University Dissertations. 1251.