Reaction of canine plasminogen with 6-aminohexanoate: a thermodynamic study combining fluorescence, circular dichroism, and isothermal titration calorimetry.
The thermodynamics of the binding of 6-aminohexanoate (6-AH) to dog glu-plasminogen has been studied. Fluorescence titrations revealed four binding sites. Three yielded positive fluorescence changes on ligand binding; one yielded a negative fluorescence change. The fluorescence data gave no indication of cooperative interactions. Binding was studied using circular dichroism (CD). Near 295 nm there were small changes associated with binding ligand. These were magnified at 235 nm, a wavelength that is mainly associated with tryptophan bands. The dissociation constants obtained from the fluorescence were applied to the CD data and fit quite well. Below 220 nm, there were no significant differences between samples with or without 6-AH and, therefore, no substantial change in the secondary structure of the protein. Isothermal titration calorimetry was used in combination with the binding constants from fluorescence to study the enthalpy and entropy contributions to 6-AH binding. The enthalpies of association for the four sites are all negative. Their absolute values are small for the tight sites and large for the weakest. -TDeltaS is negative for the tight sites and positive for the weakest. The binding of 6-AH to plasminogen is entropically driven for the two tightest sites and enthalpically driven for the weakest site. The binding of 6-AH to lys-plasminogen has been studied and differs slightly from binding to glu-plasminogen. Most importantly, the binding of 6-AH for the weak site goes from enthalpy- to entropy-driven as is found with the other sites.
Kornblatt
J A
JA
Enzyme Research Group, Concordia University, 1455 de Maisonneuve Ouest, Montreal, Quebec, Canada. krnbltt@vax2.concordia.ca
Rajotte
I
I
Heitz
F
F
D015119:Aminocaproic Acid / Q000737:chemistry*; D000818:Animals; D000933:Antifibrinolytic Agents / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D004285:Dogs; D019277:Entropy; D018082:Kringles; D010958:Plasminogen / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D017186:Titrimetry
D000933:Antifibrinolytic Agents; D010958:Plasminogen; D015119:Aminocaproic Acid