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C J Halfman

Publications and source records attributed to C J Halfman.

13 recordsLinked to original sources
PubMed2000

The relationship between the effect of lysine analogues and salt on the conformation of lipoprotein(a).

Lipoprotein(a) [Lp(a)] exhibits many of the same properties as plasminogen, owing to a similar structural makeup from a composite of multiple kringle domains. Shared behavior includes induction of an expanded conformation by lysine analogues, inhibition of this effect, and creation of a compact conformation by NaCl. Here, we examine in detail the independent and mutual effects of NaCl and 6-aminohexanoic acid (6-AHA) on the structure of Lp(a) and the relationship between the binding of the two ligands. We find that NaCl promotes the compact conformation while binding to Lp(a) homogeneously. In the absence of salt, 6-AHA leads to the complete unfolding of Lp(a), a process that is accompanied by cooperative binding. Reversal of conformation and weakening of binding occurred when one ligand was added to Lp(a) in the presence of the other, suggesting competitive binding. High concentrations of NaCl completely reversed the expansion of Lp(a) in 100 mM 6-AHA, and high concentrations of 6-AHA unfolded Lp(a) in the presence of 100 mM NaCl, but only by 30% in the case of the 15 kringle IV Lp(a) studied. Induction of the compact form of Lp(a) appears to be an effect in common with all salts examined and cannot be attributed solely to the anion, as in the case of plasminogen. The results were summarized in terms of a model of Lp(a) depicting the conformational alterations of apo(a) caused by the binding of the two ligands. In the compact conformation in NaCl, apo(a) is apposed to the particle surface. The fully expanded form in 6-AHA results from release of both the variable and constant kringle domains. In the intermediate form in water and in a solution containing both NaCl and 6-AHA, only the variable domain is released from the particle surface.

Aminocaproic Acid↗
PubMed1986

The effect of dodecyl sulfate on immunoglobulin hapten binding.

The instantaneous effect of dodecyl sulfate (DDS), in the mM concn range, on the binding of monovalent hapten by immunoglobulin was examined. Fluorescence measurements were utilized to study the effect of the detergent on sheep antiserum generated against thyroxin (T4) and against methamphetamine. Haptens were conjugated with the thiocyanate derivative of fluorescein in order to determine hapten binding on the basis of increased fluorescence polarization for the fluorescein-thiocarbamyl-hapten adducts (FT4 or FA) bound to immunoglobulin. Incubation of anti-T4-serum with DDS for 1 hr before the addition of FT4 resulted in diminished binding. The effect occurred at DDS concns greater than 0.1 mM and was essentially complete at a DDS conc of 1 mM. A kinetic study demonstrated a two stage process. An initial, rapid stage, with a half time less than 30 sec accounted for a reduction of immunoglobulin binding by 75%. The remaining 25% binding capacity was lost during a second, much slower phase with a half-time of about 11/2 hr. Prior hapten binding inhibited the effect of DDS. The degree of protection from combining site denaturation afforded by prior hapten binding was limited by the dissociation rate of bound hapten. The major, rapid phase was completely and immediately reversible by dilution. Prolonged incubation in DDS resulted in irreversible denaturation. The overall rate of DDS denaturation of the entire immunoglobulin molecule, as revealed by changes in the circular dichroism spectrum of a sheep gamma globulin fraction, was considerably slower than the denaturation rate of the combining site.

Animals↗
PubMed1986

Homogeneous, micelle quenching fluoroimmunoassay for detecting amphetamines in urine.

We developed a homogeneous fluoroimmunoassay for detecting amphetamines in urine. Only fluorescence intensity need be measured because the emission of non-protein-bound fluorescein-labeled amphetamine is preferentially quenched by detergent micelles. In a previous reported prototype assay system for measuring gentamicin in serum we used fluorescein and dodecyl sulfate (Anal Chem 1985; 57:1928-30). We have found that favorable hydrophobic and (or) ionic character of the analyte and unfavorable polar and (or) ionic character of the fluor are important determinants of the desired interactions. An anionic detergent and fluorescein, therefore, should be appropriate for apolar of cationic analytes, such as gentamicin and amphetamines. A greater [H+] at the anionic micelle surface is important for quenching emission from the fluor moiety. Millimolar concentrations of dodecyl sulfate rapidly denature immunoglobulin unless hapten is bound with sufficiently high affinity. Affinity was sufficiently high for the antibody used in the prototype gentamicin assay but not for the amphetamine antibody. Thus for the amphetamine assay, we used a non-denaturing detergent, dodecyl(oxyethylene)12 sulfate. The assay requires 30 microL of specimen in 2 mL of total assay volume. Amphetamine(d-,dl-, and meth-), at a concentration of 1 mg per liter of urine, is readily detected.

Amphetamine↗
PubMed1986

Validation of a semi-automated technique for measuring lactate in whole blood.

An electrode-based lactate analyzer was recently developed to facilitate "stat" laboratory measurement of lactate in whole blood. The results obtained with this technique were compared with those of our continuous-flow enzymatic method for 172 analyses of samples from 25 pigs; 88 analyses of samples from 12 normal human volunteers before, during, and after exercise; and 91 analyses of samples from 80 critically ill patients. This comparison revealed strongly significant linear correlations: r = 0.992 for the porcine analyses and r = 0.994 for the combined human analyses. In analytical-recovery studies with pooled porcine plasma with an initial lactate concentration of 3.85 mmol/L, 94.3% of added lactate (1, 5, and 10 mmol/L) was accounted for in the electrode technique, 96% in the reference technique. We conclude that the electrode-based lactate analyzer is rapid, precise, and accurate for measurement of lactate in whole-blood samples.

Animals↗
PubMed1982

A method for determining kinetic parameters at high enzyme concentrations.

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

Adenosine Monophosphate↗
PubMed1980

Hormonal influences on chemical carcinogenesis: studies with the aflatoxin B1 hepatocarcinoma model in the rat.

The role of adrenocorticotropin, cortisol, and corticosterone on chemical carcinogenesis was investigated using the rat aflatoxin B1 hepatocarcinoma model. The animals were divided into untreated controls and various experimental groups receiving the carcinogen alone or the carcinogen with a hormone. Animals lost during the treatment period died mostly of massive hepatic necrosis. The results following 65 weeks of observation show that: (1) hormones decrease the toxicity of aflatoxin B1; (2) adrenocorticotropin possibly exerts its influence on aflatoxin B1 hepatocarcinogenesis through adrenal stimulation, and (3) in aflatoxin B1-adrenocorticotropin treated animals, hepatocellular carcinoma, cholangiocarcinoma, and malignant lymphoma may be observed.

Adenoma, Bile Duct↗