Name:

Specific Activity at 22 °C

Category:

Activity

Property:

Specific Activity

Units:

μmol/mg/min

Measurement Technique:

Chromatography (HPLC, TLC)

Buffers:

50 mM Tris-HCl

Temperature:

22 °C

pH:

7.0

Protein Concentration:

None

Wavelength:

None

Ionic Strength:

None

Details:

Purified wild-type and variants of E. coli TK were prepared at 0.1 mg/mL, with 2.4 mM TPP, 9 mM MgCl2 and 50 mM Tris-HCl, pH 7.0. Enzymes were then incubated for 5 min by placing 100 μL samples into a water bath equilibrated at 60 or 65 °C. Sample temperatures were monitored using a digital wired-thermometer (Topac, USA) and shown to equilibrate within 5 min. Reactions were initiated by addition of pre-warmed 50 μL of 150 mM Li-hydroxypyruvate (HPA), 150 mM glycolaldehyde (GA) in 50 mM Tris-HCl, pH 7.0. Aliquots of 10 μL were quenched at various times over 120 min with 190 μL of 0.1% (v/v) trifluoroacetic acid (TFA). Triplicate reactions were analysed by HPLC. Specific activities were determined as initial rate/enzyme concentration.