Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein (I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated cells--in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-kappa B and I kappa B was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase C, cause dissociation of a cytoplasmic complex of NF-kappa B and I kappa B by modifying I kappa B. this releases active NF-kappa B which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.

https://science.sciencemag.org/content/sci/242/4878/540.full.pdf

I kappa B: a specific inhibitor of the NF-kappa B transcription factor.

http://www.jbc.org/content/275/43/33238.full.pdf

A Human Homolog of Angiotensin-converting Enzyme CLONING AND FUNCTIONAL EXPRESSION AS A CAPTOPRIL-INSENSITIVE CARBOXYPEPTIDASE

A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. The Lowry method ( Chapter 1 in vol. 1 of this series) has been widely used, but is susceptible to interference from a wide range of compounds commonly present in biological extracts. Although interference can be avoided by trichloracetic acid precipitation of the protein prior to assay, this lengthens the procedure.

The Bradford Method for Protein Quantitation

Publisher Summary This chapter discusses various methods of estimating protein concentration. Absorption spectroscopy involves the absorption of a photon by an electron. Only those photons with a certain energy level can be absorbed as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital. The peptide bond absorbs photons below 210 nm. Because of the large number of peptide bonds in a protein, this is a highly sensitive area of the protein spectrum. Although protein conformation and some absorption by tryptophan and tyrosine residues occurs in this region, less variability between proteins is observed than at 280 nm. There is a need to avoid storing buffers in plastic containers because some plastics leach plasticizers, which absorb at ultraviolet (UV) wavelengths. Detergents can also be troublesome because many absorb UV light. If the buffer or protein solution is cold, the outside of the cuvette may need to be wiped between each reading with a lint-free wiper and the readings should be made quickly after placing the cold solution into the cuvette, because atmospheric moisture may condense on the outside of the cuvette producing an erroneously high reading.

Quantitation of protein.

Measurement of protein using bicinchoninic acid

In mammals the intravenous injection of a solution of potassium chloride may produce either a sequence consisting of extrasystoles, tachycardia and finally ventricular fibrillation, or on the other hand cardiac arrest without arrythmia (5, 7, 8). Recently Nahum and Hoff (6) found in the rabbit that fibrillation was produced by the rapid injection of a concentrated solution of potassium chloride, while arrest without arrythmia occurred following slow injection of an isotonic solution. Arrest produced in this fashion is preceded by a definite sequence of electrocardiographic changes. The present studies were undertaken to determine what relationship, if any, exists between the concentration of potassium in the serum and the various electrocardiographic changes observed following intravenous injection of an isotonic solution of potassium chloride. PROCEDURE. Four dogs weighing from 15 to 20 kilos were employed in five experiments. Morphine sulphate, 10 mgm. per kgm. body weight, was injected subcutaneously thirty minutes before each experiment. A control electrocardiogram was obtained from lead II, and an initial sample of venous blood withdrawn under oil from the jugular or femoral vein. In three experiments (1, 2, 4 of table 1) an isotonic solution of potassium chloride (1.12 per cent) was then injected into the femoral vein at a rate approximating 10 cc. per minute, until the death of the animal. In one experiment (exp. 3 in table 1) injection was discontinued before cardiac arrest had occurred, and the animal allowed to recover. The same animal received at a later date an injection of an eight times isotonic solution of potassium chloride (exp. 5, table 1) which was continued until death occurred.

Electrocardiographic changes and concentration of potassium in serum following intravenous injection of potassium chloride

The toxic effects, excretion and distribution of lithium chloride

Ultrafiltration studies show that the reversible binding to bovine serum albumin of a series of barbiturates is related to the length and nature of the side chains. Binding is related to acidity, being maximal at about pH 7.8 to 8.0. With increasing drug concentration the fraction bound decreases but the total amount bound increases. With increasing protein concentration the fraction of drug bound approaches a maximum.

Thiopental is more strongly bound than comparable barbiturates and will partially displace the latter from serum albumin. Thiopental in turn may be displaced by sodium lauryl sulfonate.

Rabbit tissue homogenates are capable of binding larger fractions of barbiturate than can be accounted for by their serum albumin content. The extent of binding in vitro seems related in part to the distribution of the drugs in vivo. The extent of protein binding correlates fairly well with the known pharmacological properties of the drugs.

The data on the binding of some barbiturates to serum albumin may be represented by equations similar to those employed by others to represent the binding of other small ions by serum albumin.

The interaction of barbiturates with serum albumin and its possible relation to their disposition and pharmacological actions

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)6-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O2 affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)6-Hb, its molecular volume, O2 affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)2-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)6-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)2-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.

Paul K. Smith

Papers

Measurement of protein using bicinchoninic acid

Electrocardiographic changes and concentration of potassium in serum following intravenous injection of potassium chloride

The toxic effects, excretion and distribution of lithium chloride

The interaction of barbiturates with serum albumin and its possible relation to their disposition and pharmacological actions

Conjugation of multiple copies of polyethylene glycol to hemoglobin facilitated through thiolation: influence on hemoglobin structure and function.