Optimization of a colorimetric assay for glycosylated human serum albumin
The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.
- Research Organization:
- Univ. of Louisville School of Medicine, KY
- OSTI ID:
- 6937132
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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ALBUMINS
BIOASSAY
COLOR
FURFURAL
GLUCOSE
HYDROLYSIS
MAN
OPTIMIZATION
OXALIC ACID
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALDEHYDES
ANIMALS
CARBOHYDRATES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DICARBOXYLIC ACIDS
FURANS
HETEROCYCLIC COMPOUNDS
HEXOSES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LYSIS
MAMMALS
MONOSACCHARIDES
OPTICAL PROPERTIES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANOLEPTIC PROPERTIES
PHYSICAL PROPERTIES
PRIMATES
PROTEINS
SACCHARIDES
SOLVOLYSIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques