The incorporation of a fluorescent probe into the active sites of one- and two-chain tissue-type plasminogen activator.
Dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (DEGR-CK) was shown to inactivate both one- and two-chain human, recombinant tissue-type plasminogen activator (t-PA). The interaction of DEGR-CK with both forms of t-PA was accompanied by an identical increase in the fluorescence intensity and a blue shift in the wavelength of maximum emission, which suggests that the environment of the incorporated DEGR is similar in both one- and two-chain t-PA. The kinetics of the interaction of t-PA with DEGR-CK could be followed by both loss of activity and increase in fluorescence. The second order rate constants (k2/Ki) obtained with these two methods agreed quite well. With two-chain t-PA the values were 42 X 10(4) M-1 min-1 and 46 X 10(4) M-1 min-1 by the activity loss and fluorescence methods, respectively. With one-chain t-PA the results were 2.5 X 10(4) M-1 min-1 and 3.1 X 10(4) M-1 min-1. The rate at which one-chain t-PA is inactivated by DEGR-CK is 15 times lower than the rate with two-chain t-PA. The results demonstrated, however, that the cleavage of the one-chain protein to the two-chain form is not required for reactivity with DEGR-CK. This fluorescently labeled t-PA should be useful in probing the interactions of one- and two-chain t-PA with other proteins.
D000345:Affinity Labels; D000590:Amino Acid Chloromethyl Ketones; D001665:Binding Sites; D003619:Dansyl Compounds; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000032:analysis; D013050:Spectrometry, Fluorescence; D010959:Tissue Plasminogen Activator / Q000032:analysis*
D000345:Affinity Labels; D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D011994:Recombinant Proteins; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D010959:Tissue Plasminogen Activator