Critical role of zinc in hardening of Nereis jaws

Abstract

Hardening of invertebrate jaws and mandibles has been previously correlated to diverse, potentially complex modifications. Here we demonstrate directly, for the first time, that Zn plays a critical role in the mechanical properties of histidine-rich Nereis jaws. Using nanoindentation, we show that removal of Zn by chelation decreases both hardness and modulus by over 65%. Moreover, reconstitution of Zn yields a substantial recovery of initial properties. Modulus and hardness of Zn-replete jaws exceed those attainable by current engineering polymers by a factor of >3. Zn-mediated histidine cross-links are proposed to account for this enhancement in mechanical properties.

Fig. 1

Distribution of major inorganic components in a Nereis jaw cross section. (A) Scanning electron microscopy (SEM) image of a complete Nereis jaw. Embedded jaws were prepared by trimming to the plane indicated (red disc) with a diamond knife. Energy dispersive X-ray spectroscopy (EDS) maps of (B) untreated, (C) Tris-treated and (D) EDTA-treated jaw surfaces. Untreated and Tris-treated images are of a single jaw (EDTA treatment was done on a different jaw – distributions of Zn, Cl and Br prior to EDTA treatment were indistinguishable to those in untreated jaws). EDTA treatment, but not incubation in Tris alone, removes Zn and Cl in three distinct regions of the jaw cross section (white arrows). Zn loss is more prevalent in Br-free regions of the sample. Scale bar, 100 μm (cross sections only).

Fig. 2

Effects of EDTA treatment of jaws with uniform Br distribution. (A) Untreated, (B) EDTA-treated jaws. A minority of jaws analyzed exhibited uniform Br levels through the cross section. All other inorganic components exhibit standard distribution profiles (A). Treatment of these samples with EDTA does not lead to Zn or Cl loss. This suggests that bromination (or associated processes) may play a role in stabilizing metal coordination in the jaw matrix. Scale bar, 100 μm.

Fig. 3

Secondary ion mass spectroscopy (SIMS) depth profiles of Zn following EDTA treatment. EDTA treatment results in significant decrease of Zn in the top 16 μm of the jaw surface compared with the values observed after Tris treatment. Measurements were taken in regions that were free of Br, and Zn counts were normalized to carbon to compensate for fluctuations in signal intensity throughout the course of analysis. Signal traces for both Zn isotopes are presented.

Fig. 4

Nanoindentation of dry Nereis jaw surface. Representative load–displacement curve from nanoindentation of a prepared jaw from Nereis. Surfaces were indented with a 90° cube-cornered diamond tip at 100 μN s⁻¹ to a maximum load of 500 μN and held for 1 min. Decrease in force axis indicates viscoelastic relaxation of the sample prior to unloading. Hardness is determined as a function of the total displacement of the tip into the sample. Modulus is derived from the slope of the unloading curve.

Fig. 5

Chemical and mechanical characterization of Nereis jaws following Zn manipulation. EDS Zn maps of jaw cross sections (A) before treatment, (B) after treatment with EDTA and (C) following treatment in ZnCl₂. Indents were made following each treatment in regions indicated by the white lines (B). Both lines include indents in Zn-depleted and Zn-rich zones. (D) Hardness, H, and Young's modulus, E, are presented as a function of lateral position across the sample surface. Comparison of untreated (red line) and EDTA treated (blue line) samples indicates that both H and E are substantially decreased in regions where Zn has been removed. A significant recovery of initial H and E is observed following reconstitution of Zn in these regions (green line).

Fig. 6

Comparison of Nereis jaws to synthetic engineering polymers. Shown is a materials property chart comparing hardness H and Young's modulus E of Nereis jaws (in water) (pristine and without Zn) with those of synthetic engineering polymers. Theoretical limits for these properties are represented by the broken lines.

Fig. 7

Schematic of proposed cross-linking in Nereis jaws. Zn-mediated cross-linking of histidine-rich proteins is proposed to be responsible for enhanced hardness and modulus in Nereis jaws. Cross-linking may be intramolecular (black complexes) or intermolecular (grey complexes). Additional cross-linking via di-and/or tri-tyrosine formation is not expected to contribute significantly to mechanical properties of the bulk jaw.