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Protein chemistry of triose phosphate isomerase Burgess, Helen Diana


The protein, triose phosphate isomerase (TIM) has been isolated from fresh chicken breast muscle and purified by anion exchange chromatography on DSAE Sephadex A50 column. Further purification proceeded via Biogel A DEAE resin. The TIM fractions of both chromatographies were contained in two adjacent protein peaks, A and B. The separation of the two peaks was found to be based upon isozymic differences in the TIM active protein; isoelectric focusing, both column and gel, showed one isozyme in the Peak A protein with pI=7.65 while Peak B protein contained two isozymes of pI 7.56 and 7.49. Focusing of old peak B protein yielded a fourth isozyme with pI=7.62. No isozymic separation was observed with disc gel electrophoresis at pH 8.5. Amino acid analysis which was carried out on purified Peak A protein showed substantial deviations from literature values. The Peak A isozyme was modified via the reaction of the thiol of cysteine with the maleimide N-ethylmalelmide (NEM), or trifluoro N-ethylmaleimide (FEM), as well as with the disulfides 4,4' dithiopyridine (4-PDS) or 5,5'-dithiobis(2-nitroben-zoic acid) (DTNB). Two equivalents of reagent per molecule reacted but a kinetic non-equivalence-of the two sites to modification was observed. ¹⁹F NMR of the FEM labeled protein was performed. The final chapter of this thesis deals with the kinetic analysis at several temperatures of the modification of TIM by 4-PDS and DTNB both in the presence and In the absence of sub-strate glyceraldehyde 3-phosphate. Biphasic Arrhenius plots with a break at approximately 25°C were observed for the 4-PDS modification; In the presence of substrate, the activation energy for T>25°C was 7.2 kcal/mole while for T25°C was 4.4 kcal/mole while for T

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