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Glucuronic acid is the uronic acid corresponding to glucose; in the usual D form, carbon 6 of D-glucose carries a carboxyl group instead of its primary alcohol group. Its neutral free-acid molecular formula is ${\mathrm{C}}_6{\mathrm{H}}_{10}{\mathrm{O}}_7$. The reference carbon numbering remains that of glucose.^[ref]

Check the atom count: changing $\mathrm{C}{\mathrm{H}}_2\mathrm{OH}$ into $\mathrm{COOH}$ adds one oxygen and removes two hydrogen atoms. Loss of the acidic proton gives glucuronate, with negative electric charge. The free monomer's formula should not be substituted unchanged for a residue inside a linked polysaccharide.

In a synthesis experiment, P. L. DeAngelis used UDP-glucuronic acid together with UDP-N-acetylglucosamine as activated substrates for hyaluronan synthase. Incorporation of the two sugar types into hyaluronic acid turns a repeating structural description into a reaction whose components can be varied.^[ref]

Hyaluronan synthase builds hyaluronic acid by alternately transferring glucuronate and N-acetylglucosamine residues from activated sugar donors. The donors are UDP-glucuronic acid and UDP-N-acetylglucosamine; alternating transfers create the polymer's two-sugar repeat rather than a random mixture of the two sugars.^[ref]

For an idealized chain extension of $n$ complete repeats, the enzyme incorporates $n$ residues from each donor. Supplying only one donor cannot indefinitely produce the normal alternating chain. This makes donor omission a useful control in an enzyme assay.

In 1996, P. L. DeAngelis measured synthesis by cell-free membrane preparations from Pasteurella multocida. Activity required the sugar donors and suitable divalent ions. At the reported concentrations, $1\mathrm{mM}$ manganese produced approximately twice the incorporation observed with $10\mathrm{mM}$ magnesium. An analogous streptococcal enzyme favored magnesium instead.^[ref]

The comparison demonstrates why an enzyme name does not completely specify experimental conditions. Enzymes making the same polysaccharide can differ in ion dependence and kinetics, so an activity measured for one organism cannot automatically be assigned to every hyaluronan-producing system.

Hyaluronic acid, also called hyaluronan, is a linear glycosaminoglycan composed of alternating glucuronic acid and N-acetylglucosamine residues. It is nonsulfated, and unlike the sugar chains of conventional proteoglycans, it does not require covalent attachment to a protein core. Its carboxyl groups are largely ionized in physiological conditions, so the polymer is often encountered as hyaluronate rather than as the fully protonated acid. ^[ref]

The repeating sequence makes a useful bookkeeping example. A chain segment with $n$ complete two-sugar repeats contains $n$ glucuronic acid residues and $n$ N-acetylglucosamine residues. Its length can change enormously without changing that alternating composition. Composition alone, however, does not determine viscosity: chain length, concentration and solution conditions also matter.

Synthesis tests the two-building-block description experimentally. P. L. DeAngelis characterized the hyaluronic acid synthase from Pasteurella multocida using activated sugar donors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The enzyme incorporates the two distinct sugars into the growing chain; activity also depends on divalent ions. This turns an abstract repeating formula into a laboratory reaction whose substrates and conditions can be varied.^[ref]

Hyaluronic acid is therefore both a molecular species defined by its repeat chemistry and a material whose bulk behavior depends on its molecular population. This distinction helps explain why identifying the sugar repeat does not by itself specify the properties of every preparation.

N-acetylglucosamine is the N-acetyl derivative of glucosamine, commonly the D sugar. Its carbon-2 nitrogen carries an acetyl group, forming an amide; the open-chain reference name is 2-acetamido-2-deoxy-D-glucose.^[ref]

Compare $\mathrm{N}{\mathrm{H}}_2$ with $\mathrm{NHCOC}{\mathrm{H}}_3$. Replacing one nitrogen-bound hydrogen by an acetyl group changes the atom inventory by ${\mathrm{C}}_2{\mathrm{H}}_2\mathrm{O}$, giving neutral molecular formula ${\mathrm{C}}_8{\mathrm{H}}_{15}\mathrm{N}{\mathrm{O}}_6$. The nitrogen remains part of the amino sugar derivative even though it is no longer a free amino group.

Hyaluronic acid alternates N-acetylglucosamine with glucuronic acid. In the hyaluronan synthase experiment of P. L. DeAngelis, the activated donor UDP-N-acetylglucosamine provided this sugar type. An ideal segment with 20 complete two-sugar repeats contains 20 residues of each type and 40 sugar residues altogether.^[ref]

P. L. DeAngelis investigated the hyaluronan synthase of Pasteurella multocida, a bacterium that produces an external hyaluronic acid capsule. A 1996 study measured the enzyme's activity in cell-free membrane preparations using the activated sugar donors UDP-glucuronic acid and UDP-N-acetylglucosamine. These provide the two alternating residues of hyaluronic acid.^[ref]

Changing the available metal ions changed incorporation into the growing polysaccharide. The enzyme could use magnesium or manganese ions, but their effects differed from those reported for a related bacterial hyaluronan synthase. Thus the name of the product does not fully specify the best reaction conditions. Varying the substrates and ions turns the proposed two-sugar synthesis into a quantitative assay of an individual enzyme preparation.^[ref]