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Solid Support Synthesis of a Dnp-Labeled Peptide for Assay of Matrix Metalloproteinase-2

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Proteases in Physiology and Pathology

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Abstract

Herein, synthesis of a Dnp-labeled peptide, Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-CONH2, is described by the Fmoc solid-phase method. Post-synthesis of the peptide was purified by reversed-phase HPLC. The purity of the peptide was determined by nuclear magnetic resonance total correlation spectroscopy (NMR TOCSY), and the validity of the peptide as a specific synthetic substrate for matrix metalloproteinase-2 (MMP-2) was also assessed by measuring the specific activities of the MMP-2 using the peptide as a substrate. It was found to be a suitable substrate with respect to MMP-2 and correlated well with the [14C]-gelatin degradation assay of MMP-2. Pretreatment of the pure MMP-2 with tissue inhibitor of metalloproteinase-2 (TIMP-2), a specific endogenous inhibitor of MMP-2, prevented both the Dnp-labeled peptide substrate degradation and the [14C]-gelatin degradation. Thus, the Dnp-labeled peptide can be used as a synthetic substrate for in vitro assay of MMP-2.

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Abbreviations

APMA:

Aminophenylmercuric acetate

DIPEA:

Diisopropylethylamine

DMF:

Dimethylformamide

Dnp:

2,4-Dinitorophenyl

Fmoc:

Fluorenylmethyloxycarbonyl

HOBt:

1-Hydroxybenzotriazole

HPLC:

High-performance liquid chromatography

MBHA resin:

4-Methylbenzhydrylamine resin

MMP-2:

Matrix metalloproteinase-2

NMR:

Nuclear magnetic resonance

OPfp ester:

Pentaflurophenyl ester

PyBOP:

(Benzotriazol-1-yloxy)tris(pyrrolidino)phosphoniumhexafluorophosphate

TFA:

Trifluoroacetic acid

TIMP-2:

Tissue inhibitor of metalloproteinase-2

TNBS:

Trinitrobenzenesulfonic acid

TOCSY:

Total correlation spectroscopy

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Acknowledgments

Financial assistance from the Department of Biotechnology (DBT) (Government of India); the Council of Scientific and Industrial Research (CSIR), New Delhi; and the Indian Council of Medical Research (ICMR), New Delhi, is gratefully acknowledged. Thanks are due to Dr. Sudip Das (Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India) and Mr. Smriti Ranjan Maji (Bose Institute, Kolkata, West Bengal, India) for their expert technical help in this work.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235, West Bengal, India

    Amritlal Mandal, Tapati Chakraborti & Sajal Chakraborti

  2. Department of Physiology, University of Arizona, 1501 N Campbell Ave, Tucson, AZ85724, USA

    Amritlal Mandal

  3. Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata, 700054, India

    Atanu Maiti

  4. Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Atanu Maiti

Authors
  1. Amritlal Mandal
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  2. Atanu Maiti
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  4. Sajal Chakraborti
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Correspondence to Sajal Chakraborti .

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Editors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India

    Sajal Chakraborti

  2. St. Boniface Hospital Research Centre, University of Manitoba, Faculty of Health Sciences, College of Medicine, Institute of Cardiovascular Sciences, Manitoba, Winnipeg, Canada

    Naranjan S. Dhalla

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Mandal, A., Maiti, A., Chakraborti, T., Chakraborti, S. (2017). Solid Support Synthesis of a Dnp-Labeled Peptide for Assay of Matrix Metalloproteinase-2. In: Chakraborti, S., Dhalla, N. (eds) Proteases in Physiology and Pathology. Springer, Singapore. https://doi.org/10.1007/978-981-10-2513-6_28

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