The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry

Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.     

The reaction between hemoglobin alpha-subunit and haptoglobin.

The interaction between human hemoglobin alpha-subunit and porcine haptoglobin was investigated by polyacrylamide gel electrophoresis, gel filtration chromatography, sedimentation through excess alpha-subunit and gel filtration in an alpha-subunit-containing medium. No interaction was detected by the first two methods indicating dissociation of the complex during the application of these separation techniques. The latter two methods, in which the complex is studied in a medium of excess subunits, showed that haptoglobin became saturated with the binding of two alpha-subunits.     

Inhibition of oleic acid incorporation into a non-lipid fraction by chloroacetamide herbicides

Oleic acid is incorporated into an insoluble fraction left over after lipid extraction in Scenedesmus acutus. This incorporation is extremely sensitive to the chloroacetamide herbicide, metazachlor (I₅₀= ca 20 nM). Therefore, factors influencing the incorporation of radioactivity from oleic acid into this non-lipid fraction were investigated. S. acutus cells were cultivated under various conditions with or without inhibitors and [¹⁴C]-oleic acid was supplied to the algae; the lipids were extracted and the radioactivity incorporated in the remaining fraction monitored. The inhibition seemed specific for chloroacetamides and related classes since it was also observed with alachlor, dimethenamid and mefenacet (an oxyacetamide). In contrast, it could not be found with diuron, oryzalin, nor could it be observed with a non-herbicidal metazachlor derivative or iodoacetamide. Incorporation of oleic acid into that fraction required meta-bolically active cells and was stimulated by light. Other fatty acids (16:0, 18:2, and 18:3) were also incorporated into the non-lipid fraction but their incorporation was not inhibited by metazachlor. Among other components, the fraction contains proteins. However, a possible specific effect of chloroacetamides on the binding of oleic acid to proteins or on the in vitro activity of lipid transfer proteins could not be detected. Not much is known yet about mechanism and chemistry of oleic acid incorporation but this finding opens a new path for investigations towards the primary target of these herbicides.     

The antigen-antibody reaction in immunohistochemistry.

The problems of major concern in immunohistochemical practice are discussed in the following order: (a) the mechanism of the Ag-Ab reaction in fixed tissue as opposed to the in vitro reaction; (b) the chemistry of fixation and its influence on the final result of the immunohistochemical reaction; (c) the various procedures used for antigen retrieval in formaldehyde-fixed tissue; and (d) the consideration of the possible mechanism underlying heat-induced antigen retrieval. Suggestions for further work to attempt a clarification of the mechanism involved in the Ag-Ab reaction in immunohistochemistry resorting to existing histochemical methods for the demonstration of protein side groups are presented, together with some examples already published.     

The reaction between thiols and 8-azidoadenosine derivatives.

Thiols react at room temperature in dilute solution with 8-azidoadenosine and its nucleotides to give the corresponding 8-aminoadenosine derivatives. The reaction which takes place in the dark is base-catalysed and is particularly rapid when dithiols, e.g. dithiothreitol are used.     

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.     

The Feulgen reaction after glutaraldehyde fixation.

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH4 in 1% NaH2PO4 for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCl for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

The reaction of penicillin with proteins.

The mode of reaction of benzylpenicillin with two proteins was studied, with particular reference to the allergenicity of penicillin. These reactions, with pig insulin, and with hen's-egg-white lysozyme, were carried out in neutral solution at 37 degrees C. High concentrations of penicillin are needed to label the proteins, owing to concurrent hydrolysis of penicillin. Evidence has been obtained that the penicillin-reactive sites on the insulin molecule are the alpha-amino group at the N-terminus of the A chain and the epsilon-amino group of the lysine residue; whereas a site of reaction with lysozyme appears to be the epsilon-amino group of lysine-116.     

The ionophore-induced acrosome reaction differs structurally from the spontaneous acrosome reaction.

The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-induced vesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation.     

The reaction between some dyes and synthetic hydroxyapatite 2. Nature of the binding reaction

Synopsis In order to study the reactions involved in some of the histochemical procedures used for demonstrating calcium in calcified tissues, it was considered appropriate to use well characterized synthetic hydroxyapatite in the first instance. In the first paper of this series (Speirs, 1970), it was found that many dyes not previously used in histochemistry were capable of staining hydroxyapatite; the purpose of the present paper is to describe the numerous experimental approaches that have been made in an attempt to elucidate the mechanisms involved in the adsorption of some of these dyes by hydroxyapatite. Dyes have been grouped according to their adsorption curves (in which dye uptake by solid was plotted against the concentration of dye in solution at equilibrium). From these graphs, predictions and calculations were made concerning the orientation of the dye molecules on the surface of hydroxyapatite, the type of bonding possibly involved and the area of surface covered by each molecule. These were then related to the dimensions and structure of the dye molecules. Saturation of surface sites was achieved in the adsorption of some dyes and the nature of these sites was investigated by studying (1) competition between several dyes for the surface, (2) the accessibility of surface calcium and phosphorus in stained and unstained hydroxyapatite, and (3) the release of³²P from surface labelled hydroxyapatite during dye adsorption. Most of the dyes adsorbed from 95% ethanol were displaced relatively easily by treatment with 0.5 mM phosphate in ethanol, but those adsorbed from tris buffer, pH 7.45, were more stable when exposed to phosphate in tris. Treatment of stained hydroxyapatite with solvents containing 0.5 mM calcium reduced the rate of elution of the dyes. Convincing evidence for chelation, hydrogen bonding, ion exchange and physical adsorption processes as the mechanisms of adsorption has not been obtained. Future studies to investigate these processes are discussed.