A novel chemiluminescence (CL) biosensor for sensitive detection of wild-type p53 protein has been proposed. The wild-type p53 protein in solution was captured by highly specific consensus double-stranded (ds) oligonucleotides (ODNs) preimmobilized onto a gold plate. The cysteine residues on the exterior of the wild-type p53 molecules were then derivatized with N-biotinoyl-N\'-(6-maleimidohexanoyl) hydrazide (biotin-Mi) for the attachment of streptavidin-horseradish peroxidase (SA-HRP) complex. The attached HRP molecules could catalyze the CL reaction between luminol and H2O2, producing an enhanced CL signal. The CL intensity was dependent on the surface coverage of the HRP molecules, which was related to the concentration of wild-type p53 protein. Under the optimal experimental conditions, the CL intensity increased linearly with the concentration of wild-type p53 protein from 0.01 to 0.5nM. The detection limit was estimated to be 3.8pM. The proposed method has been successfully utilized for the assay of wild-type p53 protein in normal and cancer cell lysates. The sensing protocol is sensitive, cost-effective, and holds great promise for clinical diagnosis.

The enzyme horseradish peroxidase has many uses in biotechnology but a stabilized derivative would have even wider applicability. To enhance thermal stability, we applied consensus mutagenesis (used successfully with other proteins) to recombinant horseradish peroxidase and generated five single-site mutants. Unexpectedly, these mutations had greater effects on steady-state kinetics than on thermal stability. Only two mutants (T102A, T110V) marginally exceeded the wild type\'s thermal stability (4% and 10% gain in half-life at 50 degrees C respectively); the others (Q106R, Q107D, I180F) were less stable than wild type. Stability of a five-fold combination mutant matched that of Q106R, the least-stable single mutant. These results were perplexing: the Class III plant peroxidases display wide differences in thermal stability, yet the consensus mutations failed to reflect these natural variations. We examined the sequence content of Class III peroxidases to determine if there are identifiable molecular reasons for the stability differences observed. Bioinformatic analysis validated our choice of sites and mutations and generated an archetypal peroxidase sequence for comparison with extant sequences. It seems that both genetic variation and differences in protein stability are confined to non-helical regions due to the presence of a highly conserved alpha-helical structural scaffold in these enzymes.