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Purification and Characterization of Albumin from Frog Skin of Duttaphrynus melanostictus

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Abstract

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K i values around 1.6 × 10−7 M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.

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Abbreviations

DmA-serum:

Serum albumin of Duttaphrynus melanostictus

DmA-skin:

Skin albumin of D. melanostictus

S-2238:

h-D-Phe-Pip-Arg-pNA

BApNA:

l-Benzoyl-l-arginine-pNA

S-4760:

n-Succinyl-Ala-Ala-Ala-Pna

C-3022:

n-CBZ-Gly-Gly-Leu-pNA

BTEE:

n-Benzoyl-l-tyrosine ethyl ester

DAB:

3,3′-Diaminobenzidine tetrahydrochloride

BSA:

Bovine serum albumin

Tris:

Tris(hydroxy-methyl) aminomethane

EDTA:

Eathylene diamine tetraacetic acid

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

pNA:

p-Nitroaniline

ELISA:

Enzyme-linked immunosorbent assay

PBS:

Phosphate buffer saline

PVDF:

Polyvinylidene fluoride

HRP:

Horseradish peroxidase

DTT:

4-Dithiothreitol

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Acknowledgments

This work was supported by the grants from the National Natural Science Foundation (30700128, 30960053), and Foundation of Hainan University (hd09xm67).

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Authors and Affiliations

  1. Key Laboratory of Tropic Biological Resources, Minister of Education, Haikou, 570228, China

    Ying-Xia Zhang, Cong-Wei Chen, Ting-Ting Chi, Xing-Zhu Qi & Wen-Ting Hu

  2. Experimental Teaching Demonstration Center of Marine Biology, Hainan University, Haikou, 570228, China

    Ying-Xia Zhang, Cong-Wei Chen, Ting-Ting Chi, Xing-Zhu Qi & Wen-Ting Hu

  3. Hainan Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research (Construction Period), Hainan University, Haikou, 570228, China

    Manchuriga Wang & Shuang-Shuang Wei

  4. Haikou VTI Biological Institute, Haikou, 570228, China

    Huai Guan

  5. College of Marine Science, Hainan University, 58, Renmin Road, Haikou, 570228, Hainan, People Republic of China

    Ying-Xia Zhang

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  1. Ying-Xia Zhang
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  2. Cong-Wei Chen
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Correspondence to Ying-Xia Zhang.

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Ying-Xia Zhang and Cong-Wei Chen contributed equally to this work.

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Zhang, YX., Chen, CW., Wang, M. et al. Purification and Characterization of Albumin from Frog Skin of Duttaphrynus melanostictus . Protein J 30, 464–470 (2011). https://doi.org/10.1007/s10930-011-9349-6

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  • DOI: https://doi.org/10.1007/s10930-011-9349-6

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