Semiautomated enzymic microassay for plasma L-alanine.

Plasma L-alanine is deaminated by bacterial alanine-dehydrogenase; the resulting ammonia is dialyzed out and measured by use of a continuous flow phenol-hypochlorite colorimetric microassay. Concentrations in the range of 10-1,000 mumol alanine/l can be determined in a 50-microliter sample. The optimal conditions for the assay are specified. Study of the analytical qualities of the technique shows high specificity, good reproducibility , and a detection limit of 6 mumol/l. Usual values in human plasma from arterial or venous blood are respectively 296 +/- 166 and 376 +/- 214 mumol alanine/l (x +/- 2 SD). The usual values in rats are close to those found in man.     

An enzymatic microassay for lactose

An enzymatic microassay for lactose using a lactase enzyme derived from Saccharomyces fragilis is described. The assay uses 50-μl samples, provides 100% hydrolysis of lactose, and is sensitive within the range of 12.5–500 nmol per sample. The assay has been validated against an assay for ¹⁴C lactose which involves thin-layer chromatographic isolation of lactose. The assay is sufficiently sensitive for use in physiologic studies.     

An enzymatic-isotopic microassay for putrescine

A highly sensitive enzymatic isotopic microassay procedure for the measurement of putrescine (1,4-diaminobutane) is described. The method depends on the enhancement by putrescine of the decarboxylation of S-adenosyl-L-[carboxy-¹⁴C] methionine by baker's yeast (Saccharomyces cerevisiae) S-adenosyl-L-methionine decarboxylase in the presence of varying amounts of putrescine. The quantity of ¹⁴CO₂ evolved is a linear function of the amount of putrescine present. This method was used to measure the putrescine content of various tissues.     

Improvement and estimation of enzymic starch saccharification process

Summary The effect on the dextrose equivalent value (DE) obtained in maltodextrin saccharification with a glucoamylase preparation containing various quantities of acid stable -amylase isolated by anion exchange chromatography was investigated. When the acid stable -amylase activity was increased 2.5-fold, a maximum DE value of 95.6% (glucose content, DX, 95.0%) was reached in 48 h, 1.4 % (DX of 1.1%) higher than that of the control (DE 94.2%; DX 93.9%).     

A microassay for ATPase

A newly developed microtechnique for quantitating activity of myosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assay (ELISA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELISA plate reader. An entire 96-well microplate can be scanned in less than 2 min, and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 microliter, and the ATPase activity of as little as 10 ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique.     

A microassay for elastin

An assay for insoluble elastin is described. The procedure involves isolation of elastin by 5 m guanidine and autoclaving. The residue is solubilized with elastase and the protein released estimated colorimetrically. This assay method is sensitive in the range: 50–300 μg.     

An enzymic fluorometric micro method for determination of glycoen

A sensitive and rapid micro determination of glycogen in biological samples has been described. The method is fluoro-enzymic and is based on the conversion of glycogen to 6-phosphogluconate with the enzymes amylo-α-1,4-α-1,6-glucosidase, hexokinase, and glucose-6-phosphate dehydrogenase. The increase in NADPH is measured fluorometrically. As little as 200 ng of glycogen can be determined.     

Glucose measurement errors in enzymic starch hydrolysates at high enzyme-glucose weight ratios

The 3,5-dinitrosalicylic acid (DNS), o -toluidine, and glucose oxidase methods accurately measured concentrations of standard glucose solutions in the absence of the starch hydrolyzing enzymes Diazyme (amyloglucosidase) and Clarase (α-amylase). In the presence of high enzyme concentrations, particularly at low glucose concentrations, glucose oxidase and o -toluidine somewhat underestimated standard glucose concentrations while DNS overestimated the glucose concentration by 100%. DNS also overestimated glucose in hydrolysates of standard potato starch. Glucose recovery was estimated at almost 200% of that given by glucose oxidase when enzyme starch weight ratios were 9:1 or more. Glucose was underestimated by o -toluidine in starch hydrolysates in the presence of Diazyme at high enzyme-starch weight ratios. DNS similarly overestimated glucose in starch hydrolysates from white spruce ( Picea glauca (Moench.) Voss) and some other species, as enzyme-starch weight ratios increased. The o -toluidine and glucose oxidase reactions were more reliable. Overestimation of the DNS reaction was not improved by treating the glucose-enzyme solutions with anion or cation exchange resins or by removing the enzyme prior to measurement.     

A microassay for arylamidase activity

Greenberg's fluorimetric assay for arylamidase N activity (1) has been standardized and modified for determination of the activity in small freezedried neural lobe tissue samples (0.2–1.0 μg).     

A microassay for UDP-glucose dehydrogenase

An assay for UDP-glucuronic acid [J. Singh, L. R. Schwarz, and F. J. Wiebel, Biochem. J. 189, 369–372 (1980)] has been utilized for determining UDP-glucose dehydrogenase activity. The assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of -glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes. Unreacted 3-hydroxybenzo(a)pyrene is removed by extraction with chloroform-methanol, and the amount of gluconosylbenzo(a)pyrene formed is determined fluorometrically. Because this assay for UDP-glucose dehydrogenase is about 500 times more sensitive than spectrophotometric assays, it can be used to measure the amount of enzyme extractable from milligram quantities of connective tissue. Some kinetic properties of UDP-glucose dehydrogenase extracted from rabbit tissue have been determined. No evidence of different forms of the enzyme in rabbit liver, cartilage, or corneal stroma was found.