Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase

Abstract

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.

Simple Reversed-Phase HPLC Method with Spectrophotometric Detection for Measuring Acetaminophen-Protein Adducts in Rat Liver Samples

“…As shown in Figure 1, the recovery of APAP adduct by this method amounted to about 62% of the added amount. Although the exact reason for this deviation from maximum recovery is at present unclear, one potential contributory factor that will certainly require a more detailed investigation is the type of protease used in different laboratories to release APAP-Cys from its anchoring proteins since the activity of different types of protease can vary over a wide pH range [12]. Thus, one would expect a higher yield of APA-Cys using the present type of protease (Pronase E from Streptomyces griseus ) in a solution buffered with PBS than in one buffered with 10 mM solution of sodium acetate since the pH of the former buffer (i.e., pH 7.4) is closer to the pH declared as optimal (i.e., pH 7.5) by the supplier of this enzyme than is to that of the latter buffer (i.e., pH 6.5).…”

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase

“…One unit of proteinase K corresponds to the amount of enzyme which hydrolyses 1 pmoliliter of tyrosine or Folinactive amino acid peptide released from hemoglobin per minute at 37°C and pH 9.0 (9). The assay systems for serum AST and m-AST measurement (protease 401 or proteinase K) are the same as those used in our previous reports (5)(6)(7). Other reagent grade chemicals were obtained from Nakarai Pure Chemical Company (Osaka, Japan).…”