1. A fluorometric assay for glycosylasparaginase activity and detection of aspartylglycosaminuria
I T Mononen, V M Kaartinen, J C Williams Anal Biochem. 1993 Feb 1;208(2):372-4. doi: 10.1006/abio.1993.1063.
Recent experimental work on the mechanism of action of glycosylasparaginase suggests that the enzyme specifically reacts toward the L-asparagine or L-aspartic acid moiety of its substrates. Based on this, a new sensitive assay for glycosylasparaginase activity has been developed using L-aspartic acid beta-(7-amido-4-methylcoumarin) as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. At pH 7.5, Km = 93 microM, and as little as 1 ng of glycosylasparaginase could be detected with the assay. Hydrolysis of the substrate was inhibited by diazo-oxonorvaline, a specific inhibitor of glycosylasparaginase. In biological samples, the fluorometric assay is 40-100 times more sensitive than other published methods for glycosylasparaginase. This new assay enables a rapid enzymatic diagnosis of aspartylglycosaminuria--a genetic deficiency of glycosylasparaginase activity--with leukocyte and fibroblast samples.
2. A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy
P Ylikangas, I Mononen Anal Biochem. 2000 Apr 10;280(1):42-5. doi: 10.1006/abio.2000.4500.
The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.
3. Enzymatic diagnosis of aspartylglycosaminuria by fluorometric assay of glycosylasparaginase in serum, plasma, or lymphocytes
I Mononen, T Mononen, P Ylikangas, V Kaartinen, K Savolainen Clin Chem. 1994 Mar;40(3):385-8.
Serum, plasma, and lymphocytes from aspartylglycosaminuria (AGU) patients and carriers and from normal controls were incubated with a fluorescent glycosylasparaginase substrate, L-aspartic acid beta-(7-amido-4-methylcoumarin), and the release of 7-amino-4-methylcoumarin was measured fluorometrically after incubation for 1-4 h. The mean glycosylasparaginase (EC 3.5.1.26) activity in normal serum, plasma, and lymphocytes was 20.2 (SD 5.0) mU/L (n = 24), 17.5 (SD 5.0) mU/L (n = 24), and 242 (SD 108) mU/g protein (n = 17), respectively. The corresponding values in the Finnish AGU patients were 0.7 (SD 0.4) mU/L (n = 10), 0.3 (SD 0.3) mU/L (n = 10), and 6.0 (SD 4.6) mU/g protein (n = 7). No overlapping values were obtained between the AGU patients and the carriers in any of the samples, but the values between the carriers and controls were overlapping in 28 of 29 serum, 22 of 29 plasma, and 4 of 21 lymphocyte samples. Thus, the fluorometric glycosylasparaginase assay in various blood samples allows specific detection of the enzyme defect in AGU, but cannot be used for reliable detection of carriers of the disease.
