Analysis of the heat-induced denaturation of proteins using temperature gradient gel electrophoresis.
A new technique, temperature gradient gel electrophoresis (TGGE), was applied to the study of heat-induced protein denaturation. The gels used contained 30 mM Borax + 75 mM boric acid, pH 8.4, and various concentrations of urea. When this technique was applied to bovine serum albumin, the protein showed discontinuous bands upon melting, indicating that the thermal transition is irreversible. The apparent melting temperature, calculated based on the relative intensity of two bands in the transition region, was 58 degrees C in 2 M urea. When the thermal denaturation of bovine serum albumin was analyzed spectroscopically, the transition was again irreversible, with an apparent transition temperature of 57 degrees C, consistent with the TGGE results. Recombinant stem cell factor, recombinant granulocyte macrophage-colony stimulating factor, and catalase were also analyzed by TGGE, indicating that the technique can be used to analyze denaturation of monomeric and multimeric proteins.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Hung
L
L
Pan
V
V
Horan
T P
TP
Kolvenbach
C G
CG
Narhi
L O
LO
D000818:Animals; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000302:isolation & purification; D016298:Hematopoietic Cell Growth Factors / Q000737:chemistry / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D019089:Stem Cell Factor
D016298:Hematopoietic Cell Growth Factors; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D012710:Serum Albumin, Bovine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D002374:Catalase