(100b) Activity Measurements and Modification of Lactate Dehydrogenase Enzyme with Covalent Addition of Poly(Ethylene Glycol)

Activity Measurements and Modification of Lactate Dehydrogenase Enzyme with Covalent Addition of Poly(Ethylene Glycol)

Dyllan Rives¹, Robert Compton², Robert Chambers³

Auburn University^1,2,3

Department of Chemical Engineering

212 Ross Hall

Auburn University, Alabama, 36845-5127, USA

dmr0012@auburn.edu¹ rcompton@auburn.edu² chambro@auburn.edu³

                       A three enzyme system has been proposed in order to assist with liver metabolism and regeneration in vivo. The system is to be administered orally and will work primarily in the small intestine region of the GI tract.  The three enzymes of the system are Alcohol Dehydrogenase (ADH), Aldehyde Dehydrogenase (ALDH), and Lactate Dehydrogenase (LDH). ADH and ALDH aid in metabolizing toxins. LDH regenerates the cofactor Nicotinamide Adenine Dinucleotide (NAD+) used by both ADH and ALDH. ADH is able to rapidly convert ethanol to acetaldehyde and reduces NAD⁺ to NADH. ALDH is able to further breakdown acetaldehyde to acetate and also reduces NAD⁺ to NADH. LDH catalyzes the regeneration of NAD⁺from NADH by converting available pyruvate to lactate. LDH has been modified from its natural state by the covalent addition of Poly(ethylene glycol) (PEG). The modified enzyme has been found to have improved kinetics as well as protection from proteolytics found in the digestive tract.

                       In order to determine the change in pH range for LDH after modification, activity experiments have been performed in biological buffers ranging from pH of 4 to 9.5 in 0.5 pH increments. A UV-1800 Shimadzu UV Spectrophotometer has been used to monitor the conversion of NADH to NAD⁺by both native and modified LDH. Results of these experiments show that native LDH maintains enzymatic activity within an optimum pH range of 5 to 8. The PEG-LDH modified enzyme proved to maintain LDH’s activity within a broader pH range of 4 to 8.