Purification, Spectroscopic Characterization and o-Diphenoloxidase Activity of Hemocyanin from a Freshwater Gastropod: Pila globosa

Abstract

Hemocyanins are multi-subunit oxygen carrier proteins, found in select species of arthropoda and mollusca. Here, we have purified native hemocyanin from Pila globosa, a freshwater gastropod, verified using mass spectrometry and determined its molecular weight, secondary structure and the spectral properties, using Ultraviolet/visible, Fourier transform infra-red and Circular dichroism spectroscopy. Our results reveal the oligomeric and glycosylated nature of the protein, comprising of 400 kDa subunits, organized predominantly into a thermo-stable, alpha-helical conformation. Further, biochemical assays confirm catecholoxidase-like activity in hemocyanin, which has been used to develop a first-generation optical sensor, for the detection of phenols.

Structural insights into the interaction between molluscan hemocyanins and phenolic substrates: An in silico study using docking and molecular dynamics

“…Prior studies have reported different versions of tyrosinase-based biosensors for detection of phenols in environmental samples [33][34][35][36]. Two recent reports, one from our group [7] and the other by Raynova et al [5] described hemocyanin-based optical biosensors built by immobilizing molluscan hemocyanin on a thin film of chitosan coated on a glass slide. This first-generation sensor was able to detect catechol at concentrations of 5 ppm to 90 ppm.…”

“…However, it suffers from lower affinity for bulky phenolic substrates. Currently, optimal phenoloxidase activity in hemocyanins is obtained by either carrying out limited proteolysis [5] or pretreatment with SDS [7], prior to its immobilization on sensors. Thus, it is important to understand the structural changes caused by the action of proteolytic agents or SDS resulting in an increase in the affinity of the protein for bulky phenolic substrates.…”

“…Recent in vitro studies have detected phenoloxidase activity associated with hemocyanin suggesting its possible role in innate defence mechanisms involving the production of microbicidal pigment, melanin [4]. This activation of enzymatic properties is mediated in vitro by various agents that include proteolytic cleavage by trypsin and chymotrypsin and structural disruption by denaturants like sodium dodecyl sulphate (SDS) [4][5][6][7].…”

Evaluation of the Reproductive Performance, Body Proportions, Nutritional Value, and Biochemical Parameters of Achatina achatina

“…e unhatched eggs were broken and the state of embryonic development [7] was observed to determine the rate of early embryonic mortality (number of eggs with a dead embryo without a shell) and late embryonic mortality (number of eggs having a dead embryo with shell). At the end of the experiment, each animal of each treatment was sacrificed; the hemolymph was removed by cardiac puncture according to the method proposed by [8] to determine the total protein and cholesterol. Shell, soft tissue, pedals mass, total meat, and gonads were collected for evaluation of carcass characteristics and relative weight [9].…”

43 Studies using molecular dynamics reveal mechanism of interaction of hemocyanin with phenolic substrates

“…Hemocyanin is a multimeric, type-3 copper containing, oxygen carrier protein that exhibits phenoloxidase-like activity and is found in select species of arthropoda and mollusca. The phenoloxidase activity in molluscan hemocyanin can be stimulated either by the proteolytic removal of its C-terminal β-rich sandwich domain (Campello et al, 2008) or by denaturation with SDS (Naresh, Krupanidhi, & Rajan, 2013), both of which enhance active site access to phenolic substrates (Coates & Nairn, 2014). The mechanism of SDS-induced activation is however not well studied.…”