The catalysis of protein and nucleic acid coupling to an affinity membrane substrate.

With the model ligands studies, which included IgG, HSA, streptavidin, MEA and amine-modified DNA, it was possible to enhance the rate of covalent immobilization by using nucleophilic acylation reaction catalysts. Imidazole, triazole and 2-hydroxypyridine are readily available catalysts that are effective when immobilizing immunoglobins. 4-N,N,Dimethylaminopyridine (DMAP) as a co-reactant or as a prereactant is a potent rate enhancer with all of the molecules that were examined. The precise protocol to be used is probably best derived empirically. In addition to optimizing the amount of ligand bound or the amount of time necessary to bind a fixed quantity of ligand, it is likely that the retained functionality of the ligand may be affected by the use of reaction catalysts.

Immunological and organizational heterogeneity of histone H2a variants within chromatin of cells at different stages of Friend leukemia.

We have used antibodies directed against two histone H2a variants, H2a.1 and H2a.2, to probe chromatin structure in Friend erythroleukemia cells. Each molecule has at least one unique antigenic determinant, as well as determinants shared by both variants. Some Friend leukemia cell types contained H2a molecules which showed altered immunologic reactivity with the two antisera. The accessibility of the H2a variants in chromatin to anti-H2a antibody was different as measured by the use of whole chromatin as an immunoabsorbent and by binding of antibody to nucleosomes in a solid phase radioimmunoassay. While anti-H2a.1 IgG bound to chromatin, anti-H2a.2 IgG did not. Moreover, anti-H2a.1 IgG binding to chromatin from different Friend cell types reflected, in general, the relative amounts of H2a.1 in total chromatin. The different reactivity of the two antisera with chromatin was also observed with isolated nucleosomes: anti-H2a.1 IgG bound but anti-H2a.2 did not. Furthermore, the binding of anti-H2a.1 Ig with subfractions of nucleosomes varied; H1-depleted, high mobility group-enriched nucleosomes reacted better than H1-containing, high mobility group-depleted nucleosomes. These findings demonstrated a heterogeneity in the organization of H2a variants in chromatin within nucleosomal subfractions of chromatin and among chromatin of different Friend leukemia cell types. Moreover, most of the antigenic determinants common to both H2a variants were shown to be buried within the nucleosome core; only H2a.1-unique determinants were accessible to an anti-H2a.1 IgG molecule.

Agar gel electrophoretic determination of glycosylated hemoglobin: effect of variant hemoglobins, hyperlipidemia, and temperature.

We investigated the effect of temperature, variant hemoglobins, and hyperlipidemia on determination of glycosylated hemoglobin by an electrophoretic method (Clin. Chem. 26: 1598-1602, 1980). We found that: (a) temperature variations ranging from 4 to 30 degrees C were without effect on results obtained by electrophoresis; (b) concurrent determination of glycosylated hemoglobin by electrophoresis and column-chromatography in blood specimens from 150 diabetic patients yielded almost identical mean values for both procedures when operations were carried out at 22 degrees C; (c) electrophoretic determination of glycosylated hemoglobin in whole-blood hemolysate was not affected by concentration of triglycerides; and (d) unlike column-chromatographic procedures, which underestimate the percentage of glycosylated hemoglobin in patients with hemoglobin S and C, the electrophoretic method accurately determined the proportion of glycosylated hemoglobin in these hemoglobinopathies. Evidently, electrophoresis on agar gel is an excellent alternative to cation-exchange column-chromatographic methods for glycosylated hemoglobin.

Quantitative determination of high-density lipoprotein cholesterol by agarose gel electrophoresis.

We have developed a procedure for the determination of high-density lipoprotein cholesterol by agarose gel electrophoresis. Only 2 micro L of sample was applied to the gel. After electrophoresis at 90 V for 35 min, an enzymatic cholesterol reagent was applied. After a 30-min incubation, the high-density lipoprotein cholesterol was quantified by densitometry. Precision for this measurment approaches that reported for the heparin-manganese/Abell-Kendall method (Clin. Chem, 25: 596--609, 1979). We evaluated accuracy by comparing high-density lipoprotein cholesterol concentration measured by electrophoresis to that determined in the Framingham Heart Study procedure (J. Biol. Chem. 195: 357, 1952). The resulting correlation was excellent. By the paired Student's t-test, there was no significant difference between the two methods. The proposed method gives a linear standard curve when the concentration of total cholesterol is between 1.0 and 3.5 g/L. By accurate quantitation of high-density lipoprotein cholesterol, agarose gel electrophoresis can aid in assessment of coronary heart disease risk for a large segment of the population.

Biochemical and immunological characterization of two distinct variants of histone H2A in Friend leukemia.

Changes in the relative amount of two histone H2A subfractions have been observed in cells at different proliferative stages of Friend leukemia. Biochemical analyses of the purified H2A subfractions reveal them to be different in primary structure, and not the result of postsynthetic modifications of the same parent protein. Antibodies raised against the purified H2A.2 subfraction cross react with H2A.1 and H2A.2, but show high specificity for the immunizing subfraction at higher sera dilutions. Only H2A.2 contains a methionine which appears critical to an antigenic difference that immunologically distinguishes H2A.2 from H2A.1. The observed change in the relative amounts of two nonallelic variants of a histone coincident with changes in the physiologic states of the cell may indicate a correlation between genome structure and function.