Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 3; p. 129837
• Main Authors: Nehmé, Reine, Nasreddine, Rouba, Orlic, Lucija, Lopin-Bon, Chrystel, Hamacek, Josef, Piazza, Francesco
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2021; Elsevier
• Subjects: Animals; capillary electrophoresis; Cattle; Crowding; Dimerization; Enzyme Assays; hyaluronic acid; Hyaluronic Acid - chemistry; Hyaluronidase activity; hyaluronoglucosaminidase; Hyaluronoglucosaminidase - chemistry; Hyaluronoglucosaminidase - isolation & purification; Hydrolysis; Kinetic fragmentation mechanisms; Kinetics; Life Sciences; Male; Polyethylene Glycols - chemistry; reaction kinetics; reaction mechanisms; testes; Testis - chemistry; CE; Kinetic fragmentation mechanisms; dimer; oligomer; octamer; tetramer; Crowding; decamer; PEG; CPA; Hyaluronidase activity; BTH; HA; hexamer
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129837

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time. The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways. Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems. •A kinetic model to describe late stage degradation of hyaluronan by Hyaluronidase.•Capillary electrophoresis-based enzymatic assays used for accurate quantification.•All relevant fragmentation and transglycosylation pathways and rates identified.•Recombination of dimers to form tetramers is a dominant recombination pathway.•Macromolecular and self-crowding decrease kinetics but do not alter the mechanisms.