Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

The consequences of improper regulation of collagen turnover include diseases such as tumor cell metastasis and arthritis. Several fluorogenic triple-helical peptide (fTHP) substrates have been constructed presently to examine collagenolytic behavior. These substrates incorporate L- or D-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (Amp) or L- or D-2-amino-3-(6,7-dimethoxy-4-coumaryl)propionic acid (Adp) as the fluorophore and N-2,4-dinitrophenyl (Dnp) as the quencher. The desired sequences were C6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2. All four fTHPs formed stable triple-helices. Matrix metalloproteinase-2 (MMP-2) rates of hydrolysis for all fTHPs were considerably more rapid than corresponding MMP-1 rates. Evaluation of individual kinetic parameters indicated that MMP-2 bound to the fTHPs more efficiently than MMP-1. Comparison to a triple-helical substrate incorporating the same sequence but with a different fluorophore [Lys((7-methoxycoumarin-4-yl)acetyl); Lys(Mca)] demonstrated that the shorter side chain of Amp or Adp was better tolerated by MMP-1 and MMP-2. Adp may well be the fluorophore of choice for fTHPs, as (a) fTHPs incorporating Adp were obtained in significantly higher yields than the Amp-containing fTHPs, (b) Adp has a larger Stokes shift than either Amp or Lys(Mca) and thus has less chance of self-quenching, (c) Adp has a relatively high quantum yield, (d) the Adp/Dnp pair is compatible with multiwell plate reader formats, and (e) MMPs better tolerate Adp than Lys(Mca).

Original publication

DOI

10.1016/s0003-2697(03)00460-3

Type

Journal article

Journal

Analytical biochemistry

Publication Date

10/2003

Volume

321

Pages

105 - 115

Addresses

Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991, USA.

Keywords

Matrix Metalloproteinases, Amino Acids, Fluorescent Dyes, Circular Dichroism, Temperature, Amino Acid Sequence, Substrate Specificity, Hydrolysis, Kinetics, Thermodynamics, Molecular Sequence Data