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An amidine is a nitrogen analogue of an oxoacid derivative. In ordinary organic-chemistry usage it most often means a carboxamidine, whose central carbon is bonded to two nitrogens, conventionally represented by $\mathrm{R}-\mathrm{C}(=\mathrm{N}{\mathrm{R}}_1)-\mathrm{N}({\mathrm{R}}_2){\mathrm{R}}_3$. The IUPAC class is broader than only carbon-centred examples, so the intended structural subclass should be stated.^[ref]

A simple unsubstituted-nitrogen pattern is $\mathrm{R}-\mathrm{C}(=\mathrm{NH})-\mathrm{N}{\mathrm{H}}_2$. Choosing $\mathrm{R}=\mathrm{C}{\mathrm{H}}_3$ gives $\mathrm{C}{\mathrm{H}}_3-\mathrm{C}(=\mathrm{NH})-\mathrm{N}{\mathrm{H}}_2$ and an atom count of ${\mathrm{C}}_2{\mathrm{H}}_6{\mathrm{N}}_2$. Compare an amide, $\mathrm{R}-\mathrm{C}(=\mathrm{O})-\mathrm{N}{\mathrm{H}}_2$: replacing the doubly bonded oxygen with an imino nitrogen changes the characteristic group. Similar spelling does not make the two classes chemically identical.

Imidoesters react with amines to give amidines. This conversion connects a functional-group definition with experimental protein cross-linking, where amino groups on a protein can be modified by an appropriate reagent.^[ref] The structural pattern alone does not specify one reaction rate or one acid–base constant for every substituted amidine.

An aspartic protease is a protease whose catalytic chemistry depends on aspartic acid side chains. In common examples, their carboxyl groups position and activate a water molecule for peptide bond hydrolysis. Water supplies the attacking nucleophile, unlike a mechanism in which an enzyme side chain itself makes the initial covalent attack.^[ref]

The name concerns catalytic groups, rather than requiring an aspartic-acid residue at every substrate cleavage site. Substrate recognition and catalytic mechanism are distinct properties.

HIV-1 protease provides an experimentally characterized example. In 1989, T. D. Meek and colleagues measured apparent masses of approximately 11,000 under denaturing conditions and 22,000 under nondenaturing conditions; protein cross-linking supplied additional evidence for an active protein dimer. The ratio $22,000/11,000=2$ explains the subunit interpretation.^[ref]

The same study found pH-dependent inactivation consistent with a required unprotonated carboxyl group in the active site. Combining this chemical perturbation with cleavage activity and subunit measurements supported a catalytic classification more specifically than the enzyme's molecular mass alone.^[ref]

Dimethyl suberimidate is a bifunctional chemical reagent used for protein cross-linking. Its two imidoester ends can react with amino groups to form amidine linkages, allowing a single reagent molecule to connect two accessible protein groups.^[ref]

In 1970, G. E. Davies and G. R. Stark used the reagent to investigate subunit organization in oligomeric proteins. After cross-linking, detergent gel electrophoresis resolved species with apparent masses corresponding to integral multiples of the individual subunit mass.^[ref]

For an illustrative $20\mathrm{kDa}$ subunit, linked pairs and triples would appear near $40$ and $60\mathrm{kDa}$ before accounting for linker mass and electrophoretic behaviour. A covalent bridge can preserve an association through conditions that disrupt the original noncovalent interactions.

The observation establishes chemical proximity under the reaction conditions. It does not automatically locate a unique native interface or prove that every detected pair is a stable complex in a living cell. Reagent accessibility, possible links within one chain and collisions between molecules all affect the products. Comparing treated and untreated preparations, and identifying the protein species, makes the structural inference more informative.

G. E. Davies coauthored a 1970 method for investigating protein quaternary structure through protein cross-linking. With G. R. Stark, G. E. Davies used dimethyl suberimidate, a reagent with two reactive ends, to join nearby protein subunits before analysing the resulting products.^[ref]

SDS-PAGE resolved products with masses equal to multiples of the individual protein subunit mass. A series of linked products could therefore reveal an assembly's subunit organization after treatment that would otherwise separate its components.^[ref]

For an invented three-subunit assembly with $20\mathrm{kDa}$ per subunit, partial linking could produce species near $20$, $40$ and $60\mathrm{kDa}$. Seeing only the middle species would not establish that the original assembly contained two subunits: the completeness and selectivity of the linking reaction matter.

G. R. Stark coauthored, with G. E. Davies, a 1970 experiment using protein cross-linking to investigate the organization of oligomeric proteins. The method connected a chemical intervention to a measurable series of protein subunit masses.^[ref]

After reaction with dimethyl suberimidate, detergent gel electrophoresis separated individual chains and covalently linked combinations. The experiments included assemblies with identical subunits and assemblies containing different kinds of subunits; the latter showed unequal chemical reactivities.^[ref]

The unequal-reactivity observation gives an important interpretive control. Failure to observe a linked pair need not mean that its members were far apart in the native protein: reactive groups may have been inaccessible or poorly reactive. Chemical proximity measurements must therefore be interpreted together with the chemistry used to record them.

Protein cross-linking is formation of covalent bonds that connect sites within a protein or between different protein chains. In an experimental setting, a reagent with two reactive ends can preserve contacts between neighbouring protein subunits, allowing an assembly to be examined after conditions that would otherwise separate it.^[ref]

In a 1970 experiment, dimethyl suberimidate joined components of several oligomeric proteins. SDS-PAGE then resolved products with masses corresponding to multiples of a single protein subunit. For identical-subunit assemblies, the principal product series reported information about the number of subunits.^[ref]

For an invented example, four identical $25\mathrm{kDa}$ protein subunits can produce linked species near $25$, $50$, $75$ and $100\mathrm{kDa}$. A band near $50\mathrm{kDa}$ alone does not prove that the native assembly has only two subunits: incomplete linking can leave pairs within a larger assembly. Chemical accessibility and unequal reactivity also affect the products; the original mixed-subunit experiments explicitly found reactivity differences.^[ref]

T. D. Meek is the first author of a 1989 study characterizing HIV-1 protease produced in Escherichia coli. The purified enzyme processed a viral precursor protein into fragments consistent with the expected mature products. The investigators also compared molecular masses under denaturing and nondenaturing conditions and used protein cross-linking to probe subunit association.^[ref]

The apparent mass changed from about 11,000 to 22,000 between dissociated and associated conditions, consistent with a dimer. Cross-linking supplied a separate line of evidence for association. Pairing such structural measurements with an activity assay was important: a single chain observed under denaturing conditions need not be the functional unit of an active enzyme.^[ref]