4 research outputs found
Measuring the p<i>K</i>/p<i>I</i> of Biomolecules Using X‑ray Photoelectron Spectroscopy
Dissociation constants of GG–X–GG
and X<sub>5</sub> peptides (X = G, D, H, or K), and bovine albumin
(BSA) and fibronectin
(FN) were measured by X-ray photoelectron spectroscopy (XPS) in ultrahigh
vacuum at room temperature. The biomolecules were deposited on Au
substrates by drying 2.0 μL drops of 1.0 μg μL<sup>–1</sup> stock solutions in 100 mM sodium phosphate buffers
(pH 1–12) at room temperature. Because of the ∼+1.3
eV shift in binding energy (BE) of protonated amines, p<i>K</i> values of basic amino acids were calculated by plotting the fraction
of protonated amines as a function of solution pH. Similarly, the
BE of carboxyl groups shifted ∼−1.3 eV upon deprotonation.
While C 1s spectra were convoluted by the multiple chemical states
of carbon present in the samples, the ratio of the C 1s components
centered at BE = 289.0 ± 0.4 and BE = 287.9 ± 0.3 proved
to reliably assess deprotonation of carboxyl groups. The p<i>K</i> values for the Asp (3.1 and 2.4), His (6.7), and Lys (11.3
and 10.6) peptides, and the p<i>I</i> of BSA (4.8) and FN
(5.7), were consistent with published values; thus, these methods
could potentially be used to determine the dissociation constants
of surface-bound biomolecules
Growth and development of the barnacle <i>Amphibalanus amphitrite</i>: time and spatially resolved structure and chemistry of the base plate
<div><p>The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, <i>Amphibalanus</i> (= <i>Balanus</i>) <i>amphitrite</i> were resolved <i>in situ</i> and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide <i>ex situ</i> material identification of regions imaged by confocal microscopy. <i>In situ</i> and <i>ex situ</i> analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.</p></div
Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes
Oxidases
are found to play a growing role in providing functional chemistry
to marine adhesives for the permanent attachment of macrofouling organisms.
Here, we demonstrate active peroxidase and lysyl oxidase enzymes in
the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive
enzyme assays on isolated cement. A novel full-length peroxinectin
(AaPxt-1) secreted by barnacles is largely responsible for oxidizing
phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide
in the adhesive and oxidizes phenolic substrates typically preferred
by phenoloxidases (POX) such as laccase and tyrosinase. A major cement
protein component AaCP43 is found to contain ketone/aldehyde modifications
via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called
Brady’s reagent, of cement proteins and immunoblotting with
an anti-DNPH antibody. Our work outlines the landscape of molt-related
oxidative pathways exposed to barnacle cement proteins, where ketone-
and aldehyde-forming oxidases use peroxide intermediates to modify
major cement components such as AaCP43