J. Claude Bennett

High-sensitivity sequence determination of proteins quantitatively recovered from sodium dodecyl sulfate gels using an improved electrodialysis procedure

Abstract An improved electrodialysis procedure has been developed to recover quantitatively proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of the method has been demonstrated successfully on H-2K k , β 2 -microglobulin, complement factor D, and viral structural protein p27. The results indicate that yields exceeding 93% are obtainable, and that extended amino acid sequences of the eluted proteins in microgram quantities can be obtained in the presence of SDS without intrinsic and/or extrinsic labeling with radioisotopes.

An improved procedure for high-sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the beckman sequencer and the Ultrasphere ODS column for PTH amino acid identification

An improved procedure for high-sensitivity microsequencing is described. The method employs (i) aminoethyl aminopropyl glass beads for in situ purification of quadrol by absorbing α-amino group reactive impurities commonly found even in highly purified quadrol and (ii) an Altex 5-μm Ultrasphere ODS column and highly purified methanol which is available commercially to identify phenylthiohydantoin derivatives of amino acids by high-pressure liquid chromatography. The efficiency of the method has been demonstrated by the results of sequence analyses of peptides obtained by acid and cyanogen bromide digestions of viral proteins. A significant increase in the initial and repetitive yields has been consistently observed by this procedure.

A simple modification on the vacuum system of the Beckman automated sequencer to improve the efficiency of edman degradation

Abstract A simple modification of the high-vacuum system of the Beckman automated sequencer is described. The air cylinder controlling the high vacuum to the reaction cup has been replaced with an electrically operated stainless-steel solenoid valve. The system maintains better high vacuum without the necessity for frequent oil changes. The efficiency has been demonstrated by the results of extended amino acid sequence analysis of various unknown proteins and peptides.

Use of fluorescamine as an effective blocking reagent to reduce the background in protein sequence analyses by the beckman automated sequencer

As an effective aid to further develop the strategies for extended amino acid sequence determinations, the use of fluorescamine (fluram) as a novel blocking reagent to chemically reduce the newly generated amino termini responsible for the progressively increasing background is described. The method involves interruption of the run when proline has been determined to be the amino terminus, deletion of PITC delivery, in situ treatment of the sample with fluram within the spinning cup extraction of the reaction products using an ethyl acetate: benzene mixture, HFBA delivery, and second extraction with 1-chlorobutane. The normal program is then allowed to continue until the next proline is reached and the entire extended sequence information on nanomole amounts of proteins. The efficiency of the method has been demonstrated by direct comparison of the results of sequence analysis of amyloid P-component with (63 residues) and without (46 residues) fluram treatment.

Studies on the structural variation of the H chain of immunoglobulin M(IgM).

Abstract Four different H-chains from IgM proteins (μchains) have been studied from the standpoint of their gross molecular characteristics. These chains were shown to be homogeneous from the standpoint of sedimentation in the ultracentrifuge and migration in polyacrylamide gel disc electrophoresis. From the pattern of elution by gel filtration the molecular weight for all four calculated to be approximately 75,000. Each of the polypeptide chains had approximately 10% hexose, a C-terminal tyrosine residue, and a blocked N-terminal residue. Tryptic peptide maps suggested that the primary structure of these molecules have a significant proportion of their sequence in common, but a length of variable sequence that is limited to a perhaps relatively small part of the polypeptide chain.

Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains.

Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer

Use of o-phthaldialdehyde to chemically reduce the newly generated amino termini responsible for the progressively increasing background during an extended amino acid sequence analysis in a liquid phase sequencer has been described. The results have been compared with Fluram blocking using apomyoglobin and rabbit C-reactive protein as standard and unknown samples, respectively.

Phylogeny of immunoglobulins—purification and physico-chemical characterization of the immune macroglobulin from the turtle, Pseudemus seripta

Abstract The turtle, Pseudemus seripta , synthesizes an agglutinin antibody against Salmonella typhosa ‘H’ antigen that is confined to the macroglobulin component of whole serum. This immune macroglobulin was purified and found to have a molecular weight of 850,000. It had a total carbohydrate content of 6·7 per cent and amannose to galactose ratio different from that of human immunoglobulins. Following extensive reduction and alkylation, the molecule was dissociated into heavy and light chains which upon purification were found to have molecular weights of ≈67,000 and ≈22,500 respectively. The amino acid composition as well as certain properties of the molecule were similar to mammalian IgM. A free N-terminal residue could not be demonstrated for either the heavy or light chain. Examination by electron microscopy revealed a pentameric molecular structure. These data indicate a phylogenetic relationship among immune macroglobulins of several species and these implications are discussed.

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.

Computer-Assisted high-pressure liquid chromatography of radio-labelled phenylthiohydantoin amino acids

Abstract A computer-controlled high-pressure liquid chromatographic (HPLC) system is described to identify vitro phenyl [35S]isothiocyanate-labelled phenylthiohydantoin (PTH) amino acids from a solid-phase sequencer. Each radio-labelled amino acid from the sequencer is added to a PTH amino acid standard and the mixture separated by HPLC using a computer, programmed to detect a slope change in the absorbance. Individual fractions corresponding to the PTH amino acids are collected and counted. The sensitivity of the system is demonstrated on 700 pmoles of lysozyme.