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

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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.

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Abbreviations

UV/VIS:

Ultraviolet/visible

FTIR:

Fourier transform infra-red

CD:

Circular dichroism

KLH:

Keyhole limpet hemocyanin

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

MS:

Mass spectrometry

EDTA:

Ethylenediaminetetraacetic acid

2D-SDS-PAGE:

Two dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Acknowledgments

The authors dedicate this paper to their Beloved Founder Chancellor Bhagawan Sri Sathya Sai Baba who offers them free and value-based education at the University. We thank University Grants Commission and Department of Science and Technology, New Delhi, India for financial support. Thanks are due to Prof. D. N. Rao, IISc., Bangalore, India, for help in recording the CD spectra, Dr. Eastwood, Baylor College of Medicine for help in recording and analyzing the MS/MS data for the protein and Dr. Arun Sreekumar, Baylor College of Medicine, Houston, USA for valuable suggestions. Thanks are due to Dr. S. Venketesh, S.S.S.I.H.L, India for useful discussions.

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Authors and Affiliations

  1. Department of Biosciences, Sri Sathya Sai Institute of Higher Learning, Vidyagiri, Prasanthi Nilayam, Puttaparthy, Anantapur, 515134, Andhra Pradesh, India

    K. N. Naresh, S. Krupanidhi & S. S. Rajan

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  1. K. N. Naresh
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  2. S. Krupanidhi
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  3. S. S. Rajan
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Correspondence to S. S. Rajan.

Electronic supplementary material

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10930_2013_9490_MOESM1_ESM.tif

Alignment of sequences of peptides obtained after MS/MS analysis of hemocyanin from Pila globosa, along the entire protein sequence of hemocyanin from Enteroctopus dofleini (Protein Uniprot Accession No. O61363) (TIFF 648 kb)

10930_2013_9490_MOESM2_ESM.tif

A representative example of MS/MS spectra for the peptide sequences derived from tandem MS analysis of hemocyanin from Pila globosa. (TIFF 2316 kb)

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Naresh, K.N., Krupanidhi, S. & Rajan, S.S. Purification, Spectroscopic Characterization and o-Diphenoloxidase Activity of Hemocyanin from a Freshwater Gastropod: Pila globosa . Protein J 32, 327–336 (2013). https://doi.org/10.1007/s10930-013-9490-5

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