Quantitative and qualitative measurements of the effects of hydroxylamine on catalase activity

One of the postulated mechanism for cardioprotective potential of isoflavones is their ability to exert antioxidant action. However, various reports give conflicting results in this area. The present study was conducted with an objective to probe into the cardioprotective mechanism of isoflavones by evaluating their antioxidant potential in oophorectomized women.

Quantitative and qualitative measurements of the effects of hydroxylamine on catalase activity

When catalase is the enzyme to be inhibited, catalase inhibitors that can be used in such biological assay systems include hydroxylamine salts such as hydroxylamine hydrochloride and hydroxylamine sulfate, and sodium azide. The enzyme inhibitor can be incorporated in an integral analytic device.

It relates to methods and compositions for inhibiting catalase activity in biological Quantitative and qualitative measurements of the effects of hydroxylamine on catalase activity, and more specifically to inhibiting catalase activity in biological fluids during qualitative or quantitative analysis of biological fluids, when hydrogen peroxide participates in a signal-generating reaction.

The amount of catalase that is present in the plasma of normal human subjects can degrade hydrogen peroxide at a rate of 0. Catalase is also present in erythrocytes in levels fold higher than in plasma, giving erythrocytes the capacity to degrade gram quantities of hydrogen peroxide over several minutes, Gaecani et al.

In fact, catalase has been used to verify the specificity of assays of hydrogen peroxide in plasma or blood, and the degradation of hydrogen peroxide or inhibition of the assay system by the sample should be checked, as catalase present in blood or plasma can reduce the value of measured hydrogen peroxide to zero, Nahum et al.

The ability to measure a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, both as an adjunct to diagnosis and as therapy. To be able to perform an assay outside of a clinical laboratory setting requires that an assay have a simple protocol and be relatively free of sensitivity to small changes in the conditions under which the assay is conducted.

Although a number of systems have been developed to try to address the problems associated with analysis outside of a clinical laboratory, there is nevertheless a continuing interest in providing improved and alternative methods to those which are presently generally available.

If a clinical detection reaction requires the presence of hydrogen peroxide at any stage of the assay, any endogenous serum catalase activity present in the reaction system will interfere with the reaction.

Dilution of the sample to reduce catalase interference necessarily reduces the sensitivity of the reaction. Many clinical detection systems require the presence of hydrogen peroxide, includingdeterminations of cholesterol, triglycerides, glucose, ethanol, lactic acid, etc. For example, when free cholesterol is oxidized by cholesterol oxidase to form cholestenone and hydrogen peroxide, the hydrogen peroxide so generated can be used to measure the amount of cholesterol originally in the sample.

One of the primary needs in clinical assays is the need to determine cholesterol or triglyceride, including high and low density lipoprotein, levels in blood. There is a clear relationship between total blood cholesterol mainly the LDL fraction and coronary artery disease Journal of the American Medical Association Because cholesterol levels can be controlled by both diet and cholesterol lowering drugs for those patients at risk, the ability to monitor one's own cholesterol at home for those individuals at risk provides an important tool for monitoring cholesterol levels and thus reducing the potential for heart disease.

The ability to measure other naturally occurring compounds of physiological importance, as well as levels of synthetic drugs or hormones is also of great interest. Detection of components in liquids, such as cholesterol in blood, by test strips is well known.

Problems associated with catalase activity in human serum are avoided by including competing enzymes such as peroxidases in large excess so that the effects of catalase are minimized. In reaction systems where undiluted plasma samples are incorporated, the activity of endogenous enzymes may interfere with an analyte or precursor necessary to measure an analyte of interest.

Additionally, many enzymes which are used as reagents in clinical assays may be contaminated with catalase or with other enzymes that destabilize hydrogen peroxide. In chemical reactions that utilize hydrogen peroxide as an oxidizing agent, or which utilize hydrogen peroxide to oxidize a dye or other intermediate to generate a colored or otherwise indicator species, the stability of hydrogen peroxide is an absolute necessity to provide an accurate measurement of the analyte of interest.

Enzymes have been incorporated into detergent compositions because of the enzymes' effectiveness against various common stains which are fixed to textiles and laundry.

Quantitative and qualitative measurements of the effects of hydroxylamine on catalase activity

In particularproteolytic enzymes which possess the ability to digest and degrade protein matter are used to remove from textiles proteinic stains such as blood, perspiration, milk, cocoa, gravy and other types of sauces. In order to minimize the effect of catalase on the bleaching compounds, inhibitor compounds can be incorporated in the detergent compositions, as disclosed in Gobert, U.

The inhibitors may be contacted with the stained cloth during a soaking or prewashing stage prior to contact with the peroxide or persulfate bleaching agent or, alternatively, during the washing step, they may be included with the peroxide or persulfate bleaching agent.

Of particular importance are compositions which comprise a mixture of inhibitor and detergent. Among the inhibitors which have been found to be useful in detergent compositions for inhibiting the activity of catalase in soaking or washing solutions are hydroxylamine sulfate; hydroxylamine hydrochloride; phenylhydrazine; hydrazine; hydrazine sulfate; saturatedphenol; polyphenols substituted with at least one of NH2, S02NH2, Cl, Br, N02; aminophenols including o-amino-p-chlorophenol; aminotriazoles; alkali metal chlorates; sodium nitride; alkali metal cyanurates; and mixtures of the above.

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Many of the above inhibitors used in detergent compositions are active only at extreme pH values, such as less than 4. Additionally, many of these compounds are strong reducing agents, such as formaldehyde, which are not compatible with biological assay systems.

Others of the inhibitors which are strong oxidizing agents, including alkali metal hypochlorites, alkali metal salts of chlorocyanuric acids, or potassium salts of monopersulfuric acid, may be strong denaturing agents. These properties may be acceptable in detergent formulations, but they are completely unacceptable in clinical assay systems in which the components of the systems are to be preserved, not destroyed.

In addition, heavy metal salts such as mercuric chloride, nickel salts, or solvents such as acetone, either precipitate proteins or interfere with enzyme active site activity. Thus, many of these inhibitors cannot be used in diagnostic enzymatic reactions, in which the substances in the samples are not stable at extremes of pH or in the presence of many types of inhibitor compounds.

No admission is made that any of the background references cited above contributes prior art or pertinent prior art. It is a further object of the present invention to provide compositions for analysis of physiological fluids which are not inactivated in the reaction milieu by enzymes which decompose hydrogen peroxide.

It is another object of the present invention to provide compositions for analysis of physiological fluids which are not inactivated by catalase present in the physiological fluids.It relates to methods and compositions for inhibiting catalase activity in biological fluids, and more specifically to inhibiting catalase activity in biological fluids during qualitative or quantitative analysis of biological fluids, when hydrogen peroxide participates in a signal-generating reaction.

Apr 09,  · In a preferred embodiment, hydroxylamine salts such as hydroxylamine sulfate and hydroxylamine hydrochloride are used to inhibit catalase activity.

These compounds provide excellent inhibition of catalase activity in serum when the inhibitors are present in very low concentrations, e.g., in the range of about mM in the reaction solution. When catalase is the enzyme to be inhibited, catalase inhibitors that can be used in such biological assay systems include hydroxylamine salts such as hydroxylamine hydrochloride and hydroxylamine sulfate, and sodium azide.

Enzyme Action: Testing Catalase Activity Biology with Vernier 6A - 3 Part I Testing the Effect of Enzyme Concentration 3. Place three test tubes in a rack and label them 1, 2, and 3.

Fill each test tube with 5 mL of % H2O2 and 5 mL of water. 4. Initiate the enzyme catalyzed reaction. a. Transcript of Lab: Optimum Conditions for Catalase Activity. Conclusion Hypothesis Data Background Information Quantitative Qualitative Graph RESULT: This incongruence between our quantitative and qualitative results could in part be contributed to our method of quantitative measurement: ultimately, we were measuring the duration of the.

Since 1 molecule of catalase contains four heme groups, a molar solution of catalase would be equal to 4 M with respect to heme. Crystalline Xerum Albumin-The dry weight of a dialyzed solution of crystalline serum albumin was determined by analysis for micro-Kjeldahl nitrogen.

WOA1 - Inhibition of catalase activity in biological fluids - Google Patents