Measurement of Nonenzymatically Glucosylated Serum Protein by an Improved Thiobarbituric Acid Assay

We describe here some useful modifications of the thiobarbituric acid (TBA) assay for measurement of nonenzymatic glucosylation of serum protein. The modified assay minimizes interference by glucose without a lengthy dialysis step, and does not require an independent blank determination. These modifications should make the TBA assay more convenient for evaluating glycemic control in diabetes. Serum protein is first precipitated with cold ethanol to remove endogenous glucose. The protein is then hydrolyzed in an oxalic acid solution to release glucose as hydroxymethylfurfural (HMF). The HMF is reacted with TBA to form a chromophore which is extracted into isobutanol for spectrophotometric analysis (lambda max = 435 nm). The absorbance at 435 nm is corrected by subtracting a blank reading at 500 nm, and the nmol HMF released is determined using a standard curve prepared with pure HMF. Normal values of this assay for both adults and children are 0.38 +/- 0.10 nmol HMF/mg serum protein (means +/- 2 SD). When the assay was applied to serum samples from a group of 39 Type I diabetic children more than 90% of the children exceeded the normal range of the assay

A <i>Photinus pyralis</i> and <i>Luciola italica</i> Chimeric Firefly Luciferase Produces Enhanced Bioluminescence

We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant <i>Photinus pyralis</i> luciferase joined to the C-domain of recombinant <i>Luciola italica</i> luciferase. Compared to the <i>P. pyralis</i> enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (<i>k</i>_cat/<i>K</i>_m), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases