Development of biotinidaze enzyme activity measurement method by nuclear magnetic resonance and mass spectroscopy

Biotinidase (EC 3.5.1.12) is an enzyme with a glycoprotein structure, weighing approximately 80 kD, and is found in many tissues, especially the liver and brain. The main function of the biotinidase enzyme is to cleave and release vitamin biotin from dietary biotinyl peptide sources. In this way, free biotin, which is the cofactor of carboxylase enzymes, is provided to the system. Biotinidase deficiency, an autosomal recessive disorder, is characterized by disruption of the free biotin cycle. If free biotin cannot be provided in metabolism due to biotinidase deficiency, there is a loss of activity in biotin-dependent carboxylases (acetyl CoA carboxylase, propionyl CoA carboxylase, pyruvate carboxylase, β-methylcrotonyl CoA carboxylase). As a result, homeostasis is disrupted in important metabolic pathways such as amino acid catabolism, gluconeogenesis and fatty acid synthesis, and many diseases such as neurological disorders, developmental delays and skin diseases develop. Biotinidase, whose natural substrate is biotinylpeptides, can cleave the amide bonds between biotin and different molecules attached to it. Artificial substrates such as B-PABA and biotinyl-6-aminoquinoline have been developed to measure enzyme activity by taking advantage of this feature of biotinidase. Enzyme activity measurement methods have been developed using the spectrophotometric and fluorometric properties of these substrates. Diagnosis of biotinidase enzyme deficiency is still made using these methods in the newborn screening programs of many countries of the world, including Turkey. Unfortunately, the rate of incorrect results with the mentioned methods is very high. With the development of technology, the use of high-precision advanced technology analysis devices such as NMR and LC-MS/MS in laboratories has increased. In this thesis study, a highly sensitive, low error rate, reproducible biotinidase enzyme activity measurement method was developed using NMR and LC-MS/MS devices. During the study, domestically produced Biotinyl Aminoantipyrine (BAA) and the most commonly used commercial B-PABA molecules were used as substrates. LC-MS/MS and NMR biotinidase activity measurement method is based on the determination of the amount of biotin increased as a result of the enzyme reaction. In this context, firstly, main and fragment ions, which are biotin-specific fingerprints, were determined by LC-MS/MS. In this 5-minute method, signals characterizing biotin were detected for the main ion at 245 m/z and the fragment ion at 227 m/z. As a result of optimization studies, it was determined that biotinidase enzyme reached its highest activity in the presence of substrate, at 37 0C and in 100 mM, pH 6 phosphate buffer. In the new method, the desired level of activity could not be obtained using the BAA substrate. It was determined that the method using B-PABA as the substrate was correlated with the results obtained from external laboratories. Within the scope of the method validation study, as a result of 20 repeated measurements on two different samples, Y=0.7818 C+0.0662 and correlation coefficient R2=0.9877 were obtained. As a result of stability studies lasting one month, it was determined that the loss of activity was 15% in samples kept at +4 0C, and the loss was only 5% in samples kept at -20 0C. Measurement and evaluation experiments performed with 3 repetitions from 25 samples showed standard deviation: 0.094, LOQ: 0.939 and LOD: 0.282 IU. With this new LC-MS/MS method, more sensitive and precise results can be obtained than many biotinidase activity measurement methods currently in practice. After the protein precipitation process is completed, results can be obtained in just 5 minutes using this method. If desired, precise measurements can be obtained again even after a month from samples stored under appropriate storage conditions.