Glycyiglycyiglycineas a Synthetic Standard for Serum Protein Determination1

Glycylglycyiglycine was investigated as a reference standard for use in serum protein measurement by the biuret reaction. The tripeptide-biuret solution has a molar absorptivity of 96 at 565 nm, and absorban-ces at both 550 nm and 565 nm are proportional to concentration. By a manual reference procedure, the 550-nm absorbance of 1.0 g of tripeptide was equiv-alent to that given by 1.72 ± 0.03 g of human serum albumin or 1.43 ± 0.03 g of bovine serum albumin. By the Technicon N14b automated procedure, the absorbance of 1.0 g of tripeptide at 550 nm was equivalent to that of 1.81 ± 0.02 g of human serum albumin or 1.89 ± 0.03 g of bovine serum albumin. Results for serum protein analyses over the range 4.0 to 9.0 g/dl, when tripeptide or serum albumin was used to prepare calibration curves, showed mean differences of 0.15 g/dl in the manual mode and 0.08 g/dl in the automated mode. Peters (1) proposed that bovine serum albumin be used as a reference material for standardizing serum total protein analysis by the biuret reaction but sug-gested that an ideal standard would be a synthetic polypeptide of high molecular weight and known composition. Rappaport and Loew (2) had used a peptone solution to standardize protein analyses, but found considerable variation in biuret response from batch to batch. Malonamide, H2NCOCH2CONH2, was recommended by Hafkenscheid and Jansen (3), who used dilutions of an 0.78 mol/liter solution as standards for automated protein analyses. This re-port describes an investigation of the use of glycyl-glycylglycine, the simplest tripeptide, as a reference compound for protein analyses. Results of serum protein analyses by both manual and automated bi-uret techniques with the tripeptide as reference agree well with those obtained when human or bovine serum albumin is used for standardization