Abstract
The conditions of spectrophotometric initial rate analysis of serum alkaline phosphatase and leucine-aminopeptidase were investigated with three synthesized substrates; α-naphthylphosphate for alkaline phosphatase, L-leucyl-β-naphthylamide and L-leucyl-p-nitroanilide for leucine-aminopeptidase, respectively.
This study attempted to establish a new multi-channel automated method attached with a single light source and a detector system, for the assay of many sorts of serum enzymes including alkaline phosphatase and leucine-aminopeptidase, such as LDH, GOT, GPT, etc. which can be measured at 340 nm with coupling of NAD-NADH system.
(A) Alkaline phosphatase
(1) The results of the kinetic analysis suggest that a-naphthylphosphate would be superior as a substrate to βA-naphthylphosphate which has been used up to now.
(2) The conditions of the assay method using this substrate were as follows; a) buffered substrate, 5 mM α-naphthylphosphate in 2-amino, 2-methyl, 1, 3-propandiol. 1 M, pH 10.6. b) temperature at 37°. c) reaction mixture, buffered 0 substrate 2.9ml., serum 0.1ml. d) initial velocity was measured at 333 nm.
(3) The phenomenon observed for βA-naphthylphosphate that the optimum pH is dependent on the substrate concentration was similarly detected with this substrate.
(4) The activity ratio of α-naphthylphosphate to phenylphosphate was calculated and any different sort of sera was observed to have a different value for this ratio. This fact may offer a new concept on clinical evaluation of alkaline phosphatases which are composed of many isoenzymes.
(B) Leucine-aminopeptidase
As a substrate, L-leucyl-p-nitroanilide would be superior to L-leucyl-β-naphthylamide, in that the former was observed to have a higher molar extinction coefficient and a lower Km value than the latter.
