Fluorescence Properties and Functional Roles of Tryptophan Residues 60d, 96, 148, 207, and 215 of Thrombin

Abstract

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg 1006 ), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg 1536 ). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp 60d , however, appears to be very important for TAFI activation, and Trp 215 appears to very important for clotting and protein C activation.

Rapid Kinetics of Na+ Binding to Thrombin

“…Trp-215 in the aryl binding site has been identified as a major fluorophore responsible for the spectral change (48), but a rigorous test of the contribution of all nine Trp residues of thrombin has not been carried out. Previous studies investigated the role of Trp60d, Trp-96, Trp-148, Trp-207, and Trp-215 with Phe substitutions, but the analysis involved the activity toward thrombin substrates and not Na ϩ binding (50). Fig.…”

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

“…The reaction rate in U/mg was calculated using the molar extinction coefficient of p-nitroanilide of 8800 M −1 cm −1 at 410 nm [15]. Cuvettes were kept for at least 1 day and then measured for the exact original l-BAPNA concentration by measuring the absorbance of the liberated p-nitroanilide at 405 nm using the molar extinction coefficient of 9951 M −1 cm −1 [21] for greater accuracy. The kinetic parameters were obtained by non-linear regression, using the Briggs-Haldane modified Michealis-Menten equation.…”

Inadequate Intakes of Indispensable Amino Acids Among Homebound Older Adults

“…Threonine plays roles in mental illnesses (Sweatt et al 2003), intestinal malfunctions, and prevents fatty liver (Viviani et al 1966) caused by very low protein diets. Tryptophan is essential for the synthesis of vitamin niacin (IOM 2002), blood clotting (Bell et al 2000), and is also a major precursor of the neurotransmitter serotonin (calming effect) (Sandyk et al 1992), and the neurohormone melatonin (sleep-inducer). Fi- Dasgupta,Sharkey,and Wu 93 nally, phenylalanine is the substrate for the synthesis of tyrosine, which is utilized for the production of catecholamines (dopamine, epinephrine, norepinephrine) (Montgomery et al 2003), thyroid hormones (Lakshmanan et al 1990), and melanin.…”