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We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.

We conducted a five-component, five-level response-surface experiment to optimize the pH and the concentrations of magnesium, creatine phosphate, adenosine diphosphate, and buffer in an assay for creatine kinase. Under optimal conditions, creatine kinase activity was about 5% greater than that obtained with a previously reported assay (Clin Chem 23: 1569, 1977). We also applied a simplex maximization algorithm to the response-surface equation to locate areas of maximum sensitivity. Reaction conditions for two such areas were found, each yielding approximately 11% more activity than with the previously reported method.

We examined the stability of creatine kinase (EC 2.7.3.2) isoenzyme-3 (CK-3) in lyophilized bovine albumin matrices in the presence and absence of various sulfhydryl compounds and ADP. We initially purified CK-3 from human myocardium and skeletal muscle by the batch-chromatographic technique and by gradient elution column chromatography to specific activities of 293 and 93 kU/g, respectively. To assess stability, we subjected the lyophilized materials to storage studies at 4, 25, 37, 42, 56, and 65 degrees C and compared first-order rate constants for the decay of creatine kinase activity at 42 degrees C. Our most stable matrix contained, per liter, 2 mmol of ADP and 10 mmol of N-acetylcysteine, and had an extrapolated first-order half-life (Arrhenius plot) at -20 degrees C of approximately 60000 years.