Yashwant D. Karkhanis

A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria.

Abstract A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 n H 2 SO 4 at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.

A simplified procedure to determine tryptophan residues in proteins.

Abstract A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, 2-hydroxy-5-nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 m urea at acid pH; incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mg/ml of the protein at 280 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.

Amylopectin Synthase of Eimeria tenella: Identification and Kinetic Characterization

ABSTRACT. A soluble enzyme amylopectin synthase (UDP‐glucose‐α 1,4‐glucan α‐4‐glucosyltransferase) which transfers glucose from uridine 5′‐diphosphate glucose (UDP‐glucose) to a primer to form α‐I,4‐glucosyl linkages has been identified in the extracts of unsporulated oocysts of Eimeria tenella. UDP‐glucose and not ADP‐glucose was the most active glucosyl donor. Corn amylopectin, rabbit liver glycogen, oyster glycogen and corn starch served as primers; the latter two were less efficient. The enzyme has an apparent pH optimum of 7.5 and exhibited typical Michaelis‐Menten kinetics with dependence on both the primer and substrate concentrations. The Michaelis constants (Km). with respect to UDP‐glucose, was 0.5 mM; and 0.25 mg/ml and 1.25 mg/ml with respect to amylopectin and rabbit liver glycogen. The product formed by the reaction was predominantly a glucan containing α‐1,4 linkages. The specificity of the enzyme suggests that this enzyme is similar to glycogen synthase in eukaryotes and has been designated as amylopectin synthase (UDP‐glucose‐α‐1,4‐glucosetransferase EC 2.4.1.11).

A single-step isolation of K99 pili from B-44 strain of Escherichia coli

A single-step procedure has been developed to isolate pili from enterotoxigenic Escherichia coli strain B44 of calf origin. Pili, removed from the bacteria by heat shock treatment, were allowed to aggregate at 4 degrees C for 16 h. The precipitated pili, isolated by centrifugation, had typical pili morphology as shown by electron microscopy; ability to bind pig brush border; molecular weight greater than 6 million; and predominance of hydrophobic amino acids. On sodium dodecyl sulfate gel electrophoresis, the subunit pilin migrated as a single polypeptide of molecular weight 17,000.

Potential of the sulfobetaine detergent zwittergent 3-12 as a desorbing agent in biospecific and bioselective affinity chromatography

The isolation in our laboratories of several antigens of interest from sporulated oocysts of Eimeria species by bioselective adsorption on matrices containing immobilized antigen-specific immunoglobulins IgG was initially unsuccessful. The preparations serving as source materials for these antigens contained low levels of the zwitterionic sulfobetaine detergent, Zwittergent 3-12. Since usually immunoaffinity processes are carried out in the presence of various detergents, we were surprised, subsequently, to find this detergent to be the cause of the problem in that it prevented antigen-antibody binding. These findings led us to study the potential role of Zwittergent 3-12 as an eluting agent from matrices holding bioselectively adsorbed materials. The results of seven case studies are presented in this paper and include experiments with beta-D-galactosidase adsorbed biospecifically and bioselectively on matrices via either specific antibody or inhibitor analogue. In all cases, Zwittergent 3-12 proved to be an effective desorbing agent.

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.

Antigenic subunit of the polypeptide antigenic complex of the Melvin strain of Neisseria gonorrhoeae

Abstract An antigenic subunit of molecular weight 66,000 daltons has been isolated from the antigenic complex of the Melvin strain of Neisseria gonorrhoeae . Incubation of the complex in 8M urea at room temperature for four hours resulted in the dissociation of the subunit from the complex. It was separated from the complex by chromatography of the incubation mixture on a Sepharose 6B column in 50 mM ammonium bicarbonate pH 8.5 without 8M urea and further purified by affinity chromatography. This communication reports on a newly isolated antigenic protein devoid of LPS present in the bacteria.

Improved Sensitivity of Dabsylated Polypeptides After Polyacrylamide Gel Electrophoresis

Abstract A procedure to label polypeptides, present in complex mixtures, using dabsyl chloride (4-dimethylaminoazobenzene-4′-sul-fonyl chloride) has been described. the reaction is carried out in the presence of SDS (sodium dodecylsulfate) to avoid precipitation of polypeptides during the reaction. the dabsylated poly-peptides migrate as orange-yellow bands on SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) without change in mobility compared with undabsylated polypeptides. the sensitivity of the detection of polypeptides increases several fold when the gels are fixed in methanol/acetic acid/water (30/10/60, v/v) containing 10% TCA (w/v) (trichloroacetic acid); the dabsylated polypeptides become red and can be detected at 2 μ level. This property of visual detection of major dabsyl polypeptides in a mixture at low concentration makesthis procedure useful for the isolation of polypeptides by preparative electrophoresis.

Involvement of tryptophan in sodium dodecyl sulfate-induced conformation of K99 pilin

K99 pili from bovine strain B44 ofEscherichia coli were incubated in a solution containing 1% sodium dodecyl sulfate (SDS), 4M urea, 1% 2-mercaptoethanol (2-ME), and 10% glycerol at room temperature. After 2 weeks, there was a partial conversion of the pilin from its apparent molecular weight of 17, 000 to 21, 000 due to change in conformation as studied by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After 10–12 weeks, this change in conformation was complete. The presence of 2-ME in the incubation mixture was essential for this change; this suggested the involvement of a disulfide bond. The role of the disulfide bond could also be shown by reduction and carboxamidation of the pilin. Other modifications such as performic acid oxidation, treatment with iodoacetic acid at acid pH, and 2-hydroxy-5-nitrobenzyl bromide showed that only performic acid oxidation and HNBB modification prevented change in conformation. From these data, it was concluded that the disulfide bond and tryptophan residues of the pilin are essential for the change in conformation in SDS.