Martha H. Murtiashaw

[8] Nonenzymatic glucosylation of lysine residues in albumin

Publisher Summary This chapter describes the preparation and characterization of radiolabeled glycosylamine (GA) and N-substituted-l-amino-l-deoxyketopyranose (KP) derivatives of albumin. It also illustrates procedures for determining kinetic constants involved in their formation and dissociation and for detecting glucose adducts to lysine residues in human serum albumin (HSA) and other proteins. Nonenzymatic glucosylation is a common posttranslational modification of body protein, in which glucose reacts directly with primary amino groups on protein to yield stable covalent adducts. The sugar reacts primarily with the ɛ-amino groups of lysine residues in albumin and other proteins, although reaction at the α-amino terminus also occurs. Since, the rate of hydrolysis of glycosylamines is relatively slow at neutral pH and low temperature, the kinetics of dissociation of GA adducts can be measured by Sephadex G-25 chromatography. The Amadori adduct to the protein (after acid discharge of GA) is stable to extended incubation of the protein at 37° and has an estimated minimum half-life of about 3 weeks.

Changes in Blood Protein Glycosylation During a Diabetes Summer Camp

To evaluate changes in glycemic control during a 2-wk diabetes summer camp program, fasting plasma glucose (FPG), glycosylated hemoglobin (GHb), and glycosylated serum protein (GSP) levels were measured in a group of 36 children at the beginning and end of camp. Average FPG and GHb were unchanged during the 2-wk period, but the average decrease in GSP (7%) was significant (P < 0.005). The results of this study indicate that a measurable improvement in diabetic control occurred in some children during the 2-wk summer camp program.

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.

Albumin catabolism in diabetic rats

The kinetics of albumin catabolism were studied in normal rats and rats with streptozotocin induced diabetes (blood glucose greater than 500 mg%). Whether determined from the clearance of 125I-albumin from plasma or from the whole body, after 10 days of severe, uncontrolled diabetes there was a 30-35% decrease in the catabolic rate for albumin in the diabetic rats compared to normals. There was also about a 35% contraction of the relative extravascular distribution volume for albumin in the diabetic rats, and about a 25% decrease in the total body mass of albumin. However, the concentration of albumin in the circulation was the same in normal and diabetic animals. We conclude that when the rate of albumin synthesis is substantially depressed in diabetes, the rat maintains normal plasma albumin concentration both by decreasing albumins fractional catabolic rate and by shifting albumin from the extravascular to the vascular compartment.

A Photinus pyralis and Luciola italica Chimeric Firefly Luciferase Produces Enhanced Bioluminescence

We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis 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 (kcat/Km), 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.

Determination of the specific activity of NaB3H4

A method for the rapid determination of the specific activity of NaB3H4 is presented. NaB3H4 is used to reduce NAD+ to [3H]NADH, which is then isolated by anion exchange chromatography. The specific activity of the NaB3H4 is calculated from measurements of radioactivity and absorbance (340 nm) in the [3H]NADH fractions.