Paul K. Smith

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

Colorimetric method for the assay of heparin content in immobilized heparin preparations.

Abstract The properties of the metachromatic dye toluidine blue have been utilized to determine colorimetrically the amount of heparin covalently coupled to Sepharose. The method involves monitoring the dye depletion in the supernatant at 631 nm as Toluidine blue is adsorbed onto the heparin polymer upon the beaded matrix. The procedure represents a simple assay technique which allows the direct quantitation of heparin in immobilized heparin preparations.

Preparation and Use of a Boronic Acid Affinity Support for Separation and Quantitation of Glycosylated Hemoglobins

Abstract Cross-linked agarose activated with carbonyl diimidazole and subsequently coupled to aminophenyl boronic acid provides a highly efficient matrix for separation and quantitation of glycosylated hemoglobins The method described is highly reproducible. rapid and flexible in that it can be adapted to column or batch formats The method can tolerate moderate fluctuations in temperature and pH of buffeused without significantly altering results Hemoglobin fractions separated by this affinity support were Subjected to isoelectric focusing which revealed a heterogeneous population of glucose modified proteins.

Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.

A new cleavable reagent for cross-linking and reversible immobilization of proteins☆

Abstract We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.

N-(β-iodoethy)trifluoroacetamide: A new reagent for the aminoethylation of thiol groups in proteins

Abstract β-Iodoethyltrifluoroacetamide was prepared and evaluated for use in the aminoethylation of sulfhydryl groups in proteins. Lysozyme, ribonuclease A, chymotrypsinogen A, and soybean trypsin inhibitor were reduced using dithiothreitol in 6 m guanidine hydrochloride. The reduced proteins were then treated with the reagent. Conversion of cysteinyl to aminoethylcysteinyl residues averaged 97%; no destruction of nontarget residues was observed.

Clinical Use and Time Relationship of Changes in Affinity Measurement of Glycosylated Albumin and Glycosylated Hemoglobin

Simple techniques for measurement of glycosylated hemoglobin and glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns have recently been developed. This study explored the time course of changes in glycoalbumin versus those of glycohemoglobin in response to rapid changes in ambient glucose concentration. One would predict that glycoalbumin levels would change more rapidly than glycohemoglobin levels due to the shorter half-life of albumin than hemoglobin. This was found to be the case in a group of rabbits rendered diabetic with alloxan. Glycoalbumin levels plateaued 4 weeks after alloxan administration, while glycohemoglobin levels were still rising. In a group of diabetic patients in whom glucose levels were initially poorly controlled, strict diet or intensive insulin management were used to rapidly bring glucose levels under control. In this group of patients, the glycoalbumin values entered the normal range and plateaued, while glycosylated hemoglobin levels were still falling. Glycoalbumin determination by affinity chromatography is a valuable adjunct to glycosylated hemoglobin determination in evaluating near term control of blood sugar values.

Synthesis and characterization of N-(4-azidophenylthio)phthalimide: A cleavable, photoactivable crosslinking reagent that reacts with sulfhydryl groups

Abstract The cleavable, photoaffinity label precursor, N -(4-azidophenylthio)phthalimide has been synthesized and purified. The recrystallized product was identified by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The compound modifies free thiol groups to yield the corresponding S -4-azidophenylthio derivatives. In order to examine the biological applications of this compound, yeast iso-1-cytochrome c , containing a single free cysteine residue, was modified and characterized. The 102- S -(4-azidophenylthio)-iso-1-cytochrome c was found to contain 1 mol of label/mol of cytochrome c . The photoaffinity labeling of purified, detergent-solubilized yeast cytochrome c oxidase was examined. Photolysis products of crosslinking could be analyzed on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The crosslinked products were readily cleaved by dithiothreitol. The use of this compound as a sulfhydryl-specific photolabile, bifunctional crosslinking reagent is discussed.

Comparison and contrast of affinity chromatographic determinations of plasma glycated albumin and total glycated plasma protein

Techniques for affinity measurement of glycated albumin and for glycated total plasma protein have been developed. The two techniques were contrasted. Both techniques are linear over a 100-fold range of sample concentrations. There appears to be a non-specific early glucose binding phase to non-albumin plasma proteins. Although this phase is detected by radioactive incorporation and thiobarbituric acid, it does not interfere with the affinity determination, which does not appear to detect the early binding species. The correlation of glycated albumin levels with glycated hemoglobin levels is much stronger than that of glycated globulin levels with glycated hemoglobin levels. Due to the large contribution of glycated albumin levels to total glycated serum protein levels, the correlation of the latter with glycated hemoglobin levels is sufficiently strong to allow the use of either technique as an adequate index of glycation.

An imidoester spin probe of membrane protein interactions: Application to cytochrome c

Abstract The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10 −9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10 −9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10 −9 ). Cytochrome c -oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.