TY - JOUR
T1 - Analysis of matrix metalloproteinase triple-helical peptidase activity with substrates incorporating fluorogenic L- or D-amino acids
AU - Lauer-Fields, Janelle L.
AU - Kele, Péter
AU - Sui, Guodong
AU - Nagase, Hideaki
AU - Leblanc, Roger M.
AU - Fields, Gregg B.
N1 - Funding Information:
We thank Drs. Frank Marı́ and W. Craig Byrdwell for their assistance with the electrospray mass spectrometric analyses. We gratefully acknowledge support of this work from the National Institutes of Health (AR 39189 to H.N., CA 77402 and CA 98799 to G.B.F.), the Wellcome Trust (Reference No. 057508 to H.N.), and the FAU Center of Excellence in Biomedical and Marine Biotechnology.
PY - 2003/10/1
Y1 - 2003/10/1
N2 - 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 C 6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly∼ 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).
AB - 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 C 6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly∼ 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).
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U2 - 10.1016/S0003-2697(03)00460-3
DO - 10.1016/S0003-2697(03)00460-3
M3 - Article
C2 - 12963061
AN - SCOPUS:0042696223
VL - 321
SP - 105
EP - 115
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -