Method for microassay of microsomal heme oxygenase activity

An isotope dilution technique for the estimation of microsomal heme oxygenase activity is described. The method is based on the measurement of bilirubin-¹⁴C formation from hemin-¹⁴C in the presence of excessive NADPH-dependent biliverdin reductase. The procedure is specific and sufficiently sensitive for use in the measurement of microsomal heme oxygenase activity in needle biopsy specimens.     

A microassay for the determination of monoamine oxidase activity using electron capture gas chromatography

A sensitive and specific assay for determining monoamine oxidase (MAO) activity in serum and platelets is described. The procedure employs m-iodobenzylamine as substrate. The product, m-iodobenzaldehyde, is separated on an OV-17 column and measured by electron capture. Gas chromatography offers the advantage of analytical specificity without the need for additional procedural steps to eliminate potential interferences. Electron capture detection of the iodinated aldehyde is sufficiently sensitive to allow routine analysis on platelet samples of less than 15 μg of protein and serum aliquots of 50 μl or less. Analysis of approximately 80 samples per day may be accomplished by a single worker by employing an automatic sampling system for gas chromatographic injection. This fact, in addition to the small sample size, makes the method particularly suitable for the determination of large numbers of clinical samples.     

A microassay for lysyl oxidase activity.

Pinnell and Martin's [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.     

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 proteolytic activity

A quantitative procedure for measuring proteolytic activity, utilizing azoalbumin as substrate, has been developed for use in microtiter plates. An enzyme-linked immunosorbent assay reader is used to measure absorbance. The procedure is sensitive, as well as being both rapid and economical. It is particularly convenient for measuring large numbers of samples, such as fractions from column chromatography.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

A radiometric microassay for cellulase activity

The method described measures the release of ¹⁴C products from [U-¹⁴C]cellulose. The assay is simple and utilizes very small incubation volumes. Activity is evident within the first 30 min of reaction. This method should be particularly suited to activity surveys of culture filtrates from fermentations as well as the analysis of fractions from enzyme purification studies.     

Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity.

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.     

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.     

A new fluorescent microassay method for pepsin using succinyl-albumin.

A method was developed for fluorescent microassay of pepsin with a fluorescent reagent, fluorescamine, and a nonquenching substrate, succinyl-albumin. In this method hydrolysis of succinyl-albumin by pepsin at pH 2,0 was stopped by adding phosphate buffer, pH 6.1, and newly liberated amino groups in the reaction mixture were determined quantitatively by fluorescence after adding fluorescamine. Fluorescence increased linearly with 1.0 to 18 ng of hog pepsin. The assay was 200 times more sensitive than the modified micromethod of Anson [(1939) J. Gen. Phys.22, 79–89].     

Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O(2) binding. Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe-O-O angle is 110 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O-O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.     

Rapid assay for nitric oxide synthase using thin-layer chromatography

A simple, sensitive, and rapid method to determine the nitric oxide synthase (NOS) activity in crude cell extracts has been developed. The method takes advantage of differential migration of arginine and citrulline on silica gel thin-layer chromatography (TLC) with the specified buffer system. We have shown that products obtained by treating [14C]arginine with crude mouse hippocampal homogenate can be separated by methanol precipitation followed by TLC. The separated products of the enzyme reaction can be quantitated by radiometric scanning of the TLC plate or by counting in a scintillation counter. Inhibition of conversion of l-arginine to l-citrulline by NG-monomethyl-l-arginine acetate, a specific inhibitor of NOS, confirmed the NOS assay described in this investigation. This method is versatile and allows rapid simultaneous assay of several samples in a short period of time. Therefore, this assay is very useful for both qualitative and quantitative estimation of NOS activity.