Shinichi Teshima

Determination of α-amylase in biological fluids using a new substrate (β-2-chloro-4-nitrophenyl-maltopentaoside)

Abstract A novel substrate, β-2-chloro-4-nitrophenylmaltopentaoside(βCNPG5), was used for the enzyme-coupled determination of α-amylase in biological fluids. It was hydrolyzed specifically by α-amylase to about 90% producing β-2-chloro-4-nitrophenylmaltoside (βCNPG2) and maltotriose. Under the assay conditions, the absorption of 2-chloro-4-nitrophenol (CNP) generated by the secondary reaction of α-glucosidase and β-glucosidase as auxiliary enzymes is about twice the absorption of 4-nitrophenol (PNP), which is the end product currently measured in some α-amylase assay methods. The sensitivity of the assay using βCNPG5 was thus much higher than that using 4-nitrophenyl-maltopentaoside (PNPG5) as substrate. The absorption of CNP did not fluctuate with temperature or with pH between 6.8 and 7.2, which are the conditions normally used for determination of amylase activity in biological fluids.

Determination of α-amylase using a new blocked substrate (3-ketobutylidene β-2-chloro-4-nitrophenyl-maltopentaoside)

Abstract A new substrate, 3-ketobutylidene β-2-chloro-4-nitrophenylmaltopentaoside (3KB-CNPG5), was used for the determination of α-amylase (EC 3.2.1.1) in serum and urine. Under this α-amylase assay condition, 3KB-CNPG5 is resistant to glucoamylase and α-glucosidase, which are auxiliary enzymes, because the 4- and 6-positions of the non-reducing-end glucose residue are modified by the 3-ketobutylidene group. The assay using 3KB-CNPG5 for α-amylase activity is a highly sensitive method that uses 2-chloro-4-nitrophenol (CNP) as an aglycone, and is a stable method for determination of α-amylase activity in biological fluids.

Determination of acid phosphatase in biological fluids using a new substrate, 2,6-dichloro-4-nitrophenyl phosphate

A new substrate, 2,6-dichloro-4-nitrophenyl phosphate (DCNP-P), is used for the determination of acid phosphatase (EC 3.1.3.2) in serum and urine. It was hydrolyzed by acid phosphatase to 2,6-dichloro-4-nitrophenol (DCNP) and phosphoric acid. At a pH of 4.5-6.0, the absorption of DCNP liberated by acid phosphatase was much higher than that of p-nitrophenol, which is commonly used as an aglycone in the acid phosphatase assay. By using DCNP-P as a substrate for acid phosphatase activity, determinations can be made without the colour reaction which requires the addition of an alkaline solution, and can be determined by the rate assay that does not require measurement of sample blanks in serum or urine. This method using DCNP-P is highly sensitive and is the most suitable for the rate assay of acid phosphatase activity in biological fluids.