Yoshihide Shimabayashi

Investigation of methods of enzyme immobilization on a coated-wire electrode for the development of micro-biosensors

Abstract Tridodecylamine- and carboxyl-substituted poly(vinyl chloride)-based hydrogen ion-selective coated-wire electrodes are investigated as transducers for micro-biosensors. Various methods for immobilization of enzyme on the electrode were evaluated by using penicillinase as a model enzyme. The best method was immobilization with glutaraldehyde on the electrode previously modified with bovine serum albumin in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to increase the amount of immobilized enzyme. A stable, large potential change is obtainable even in salt-containing samples with this sensor. The possibility of the practical use of such a micro-biosensor is suggested.

Application of a coated-wire electrode to an acetylcholine sensor

Abstract An acetylcholine sensor was constructed with acetylcholinesterase which was immobilized on a hydrogen ion-selective coated-wire electrode and fundamental properties of this sensor were investigated. Acetylcholine could be determined in the range 0.1–10 rum with response times of 3–10 min. The effects of pH and concentration of buffer solution on the determination and fluctuations in the data obtained with this sensor were also investigated. Possibilities for the practical use of this acetylcholine micro-sensor are suggested.

FINDINGS OF TRIGONELLINE DEMETHYLATING ENZYME ACTIVITY IN VARIOUS ORGANISMS AND SOME PROPERTIES OF THE ENZYME FROM HOG LIVER

Trigonelline demethylating enzyme activity was found widely in animals, plants and microorganisms. Very high enzyme activity of this enzyme was detected in hog liver. Properties of the hog liver enzyme were investigated. Optimum pH for the enzymic reaction was observed at 8.5. The Km value for trigonelline was calculated at 2.77 mM. Addition of any cofactor is not required for the reaction The enzyme activity was inhibited by heavy metal ions. The reaction product was identified as nicotinic acid. Proposed enzyme reaction mechanism and the role of this enzyme in biosynthesis and metabolism of NAD are discussed.

Biosensor for peptide determination constructed by immobilizing proteolytic enzymes on coated-wire electrodes

Abstract A biosensor for peptide determination was constructed with trypsin or α-chymotrypsin immobilized on hydrogen ion-selective coated-wire electrode and its fundamental properties were investigated with two artificial peptides. The substrates could be determined in the range 0.1–40 mM with response times of 5–10 min. The effects of pH and concentration of buffer solution on the determination were investigated.

A new fluorometric assay method for quinolinic acid

A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215-220 degrees C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assay method for quinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assay method may be practically applicable.

Deoxyribonuclease of Chick Embryo: Purification and Characterization

A deoxyribonuclease (DNase) was purified approximately 200-fold from 14-day-old chick embryos. The purified DNase did not contain detectable phosphomonoesterase, phosphodiesterase or ribonuclease activity, and it was homogeneous on gel electrofocusing and gave a single band of protein on polyacrylamide gel electrophoresis. The enzyme was activated slightly by Mg2+, inhibited completely by Hg2+ and moderately by Cu2+, had a pH optimum at 5.3, a molecular weight of 37,000 and an isoelectric point of 6.5. Its mode of action was primarily endonucleolytic, yielding oligonucleotides ending in 5′-phosphates, and a preference was shown for native DNA as substrate.

On a Dexyribosyl Compound in the Ribonucleic Acid Preparation

Ribonucleic acid prepared according to modified Kirby’s phenol method from 14 day old chick embryos was fractionated into three peaks by MAK column chromatography. To the peak eluted in the range from 0.65 to 0.7 m NaCl, the name “Peak II” was given tentatively. Purified Peak II was homogeneous upon ultracentrifugation, polyacrylamide gel electrophoresis and elution patterns of chromatographies.The peak gave a positive result with the orcinol reaction. Furthermore, the hydrolyzate by DNase and snake venom was active in promoting the growth of L. leichmannii 7830. The peak was not disrupted by treatment with RNase, but was with DNase. Peak II treated with heat gave the same fractionation profile as the native peak by gel filtration on Sephadex G–200.By treatment with 0.3 n KOH at 37°C for 18 hr, Peak II was resolved into two peaks, Peak II–1 and Peak II–2. It was found that Peak II consisted of the susceptible and the non-susceptible components to the alkali. From the susceptible component (Peak II–2) the ...

Biochemical Studies during the Development of the Chick Embryo: Part IV Determination of Deoxyribosides in the Acid Soluble Fraction of the Chick Embryo and those Changes with the Incubation

The presence and the change of deoxyribosidic compounds in the acid extract of the embryo with the incubation were examined with an aid of the organism, L. leichmannii. The main deoxyribosidic compounds in the extract prepared from the 18 th day embryo were identified as uracil, cytosine and thymine deoxyribosides and deoxyribotides of cytosine and thymine from the behaviour on paper chromatographic and paper electrophoretic separation. A small amount of purine deoxyribosyl compound which was assumed as hypoxanthine deoxyriboside was detected, and the content of which per 1 g of fresh embryo changed with the lapse of the incubated day; especially, the content was minimum at the period from the 10 th to 15 th day incubation. At this period, the total growth promoting compounds contained 50% of deoxyribotide though deoxyribosides was lower than that of the other days. This period is the most significant stage of the embryo growth and the most active time of synthesis of DNA through the embryo growth.

Biochemical Studies During the Development of the Chick Embryo: Part I. Dynamic Changes of Nucleic Acids and Vitamin B12

The days when the concentration of conjugated- and free- vitamin B12 per wet weight g increased or decreased agreeded with that of nucleic acids in the chick embryo from 3rd through 18th day of incubation. It is intereresting that the day of increasing or decreasing of those compounds concentration corresponds with the important stages through the growth of embryo. The changes of these compounds in the egg contents free of embryo were estimated. Although a considerable large amount of free vitamin B12 was contained in the embryo, the most vitamin B12 in the egg content except the embryo existed in the conjugated state. It seems that the amount of total vitamin B12 in the egg during the incubation is relatively constant.