On-line procedures for alkylation of cysteine residues with 3-bromopropylamine prior to protein sequence analysis.

We have previously shown that 3-bromopropylamine offers several advantages over other alkylating reagents in the modification and subsequent identification of cysteine residues by protein sequencing. We describe here simple on-sequencer procedures for alkylating cysteines in proteins which employ the reduction of cystines in proteins with tri-n-butylphosphine and concomitant alkylation of the resulting cysteines with 3-bromopropylamine. Addition of an aqueous acetone wash to a modified reaction cycle on the Applied Biosystems 477A sequencer removes excess 3-bromopropylamine. As a result, very little background in the first step of the sequence analysis is seen. Under these conditions, cysteines are readily modified and identified during sequencing. Moreover, very little preview of the next amino acid is observed, which indicates that the N-terminal amino acid is not appreciably alkylated by 3-bromopropylamine. On-sequencer methods have been developed for proteins spotted onto glass fiber filters and proteins electroblotted onto polyvinylidene difluoride membranes.

Production and in vivo characterization of a bifunctional antibody (IVA039.1) with specificity for the mouse interleukin-2 receptor and vinca alkaloids.

The autoreactive T cell plays a pivotal role in the pathogenesis of type I diabetes in humans and in rodent animal models. Elimination or attenuation of these cells may provide a means to treat the disease. The use of antibodies directed to T cells has shown varying degrees of effectiveness in the treatment of autoimmune disease. The use of a bifunctional antibody directed to T cells with a cytolytic agent may provide an additional level of therapeutic efficacy compared to anti-T-cell antibodies alone. To test this hypothesis, we prepared a bifunctional antibody (IVA039.1) with specificity for the mouse interleukin-2 (IL-2) receptor and vinca alkaloids. The antibody was derived from the fusion of vinca immune spleen cells with PC61 5.3, a hybridoma that produces rat anti-mouse IL-2 receptor antibody. IVA039.1 was purified by affinity chromatography through Protein A and anti-vinca affinity columns followed by TSK-DEAE high-pressure liquid chromatography (HPLC). Bifunctionality of the antibody was confirmed by fluorescence-activated cell sorting (FACS) analysis, enzyme-linked immunoadsorbent assay (ELISA) and a cell assay designed to measure simultaneously both IL-2 receptor and vinca reactivities. The biodistribution of IVA039.1 was determined in normal and streptozotocin-complete Freund's adjuvant (CFA) induced diabetic mice. Enhanced uptake of IVA039.1 was observed in the pancreata, spleens, and lymph nodes of diabetic compared to normal mice. These data suggest that bifunctional antibodies that can deliver cytolytic agents to T cells may be appropriate candidates for the treatment of diabetes and other autoimmune diseases.

Quantitation of cysteine residues alkylated with 3-bromopropylamine by amino acid analysis.

A new versatile reagent, 3-bromopropylamine, for the quantitative analysis of cysteine residues in proteins and peptides is reported. When added to amino acid standards, the 3-bromopropylamine derivative of cysteine, S-3-aminopropylcysteine, elutes in a unique position on four different amino acid analysis systems without modification to their standard gradients. Optimized conditions for the complete alkylation of cysteines in proteins with 3-bromopropylamine are described. The S-3-aminopropylcysteine is stable to standard acid hydrolysis conditions used for amino acid analysis. Cysteine values are within 10% of the predicted value in the amino acid analysis of acid hydrolysates of known proteins based on quantitation with S-3-aminopropylcysteine. No evidence of alkylation of other amino acids by 3-bromopropylamine is apparent from the amino acid analysis of proteins alkylated under the optimal conditions. These results expand the application of 3-bromopropylamine to include quantitation of cysteine by amino acid analysis as well as the previously reported identification of cysteines by protein sequencing.

How the anti-(metal chelate) antibody CHA255 is specific for the metal ion of its antigen: X-ray structures for two Fab'/hapten complexes with different metals in the chelate.

Antibodies with bound metal-chelate haptens provide new means for exploiting the diverse properties of metallic elements. The murine monoclonal antibody CHA255 (IgG1 lambda) binds the metal-chelate hapten indium (III)-4-[N'-(2-hydroxyethyl)thioureido]-L-benzyl-EDTA (designated In-EOTUBE) with high affinity (K(a) = 1.1 x 10(10) M-1). Antibody binding is highly specific for the indium chelate; the affinity decreases as much as 10(4) with other metals, even those having ionic radii close to indium. To better understand this selectivity, the crystal structure of the antigen-binding fragment (Fab') of CHA255 complexed with its hapten, In(III)-EOTUBE, was determined by molecular replacement and refined at 2.2-A resolution. The structure of CHA255 Fab' complexed with Fe(III)-EOTUBE was also determined and refined at 2.8-A resolution. In both structures, the hapten's EDTA moiety is half-buried near the center of the complementarity-determining regions (CDR's). Five of the six CDR's on the Fab' interact with the hapten through protein side-chain atoms (but not main-chain atoms). A novel feature of the In-EOTUBE/Fab' complex is coordination of the indium by N epsilon of one histidine from the heavy chain's third CDR (distance = 2.4 A). The histidine coordination is not observed in the Fe-EOTUBE/Fab' complex, due mainly to a slightly different hapten conformation that reduces metal accessibility; this may partially explain the 20-fold lower affinity of CHA255 for iron hapten. An unexpected feature of the Fab' overall is an elbow angle of 193 degrees (the angle between the pseudodyad axes of the Fab's constant and variable domains).

Identification of cysteine residues alkylated with 3-bromopropylamine by protein sequence analysis.

A new reagent for the routine identification of cysteine residues during protein sequencing is described. This method employs 3-bromopropylamine to alkylate cysteines in proteins after reduction with dithiothreitol. Upon sequencing of the protein on an Applied Biosystems 477A protein sequencer, the aminopropylcysteine residue yields a phenylthiohydantoin (PTH) derivative which elutes reproducibly at a unique position immediately after PTH-leucine; baseline resolution is achieved without modification of the PTH analyzer gradient. Unlike PTH-pyridylethylcysteine, the relative elution position of the aminopropylcysteine PTH derivative is not affected by changes in the ionic strength of the analyzer solvents. In addition, the previewing of the next amino acid which is observed in proteins modified with 4-vinylpyridine does not occur in aminopropylated proteins. Preparation of alkylated proteins for electroblotting onto polyvinylidene difluoride (PVDF) membranes and methods for desalting alkylated proteins immobilized on precoated glass fiber filters or PVDF membranes are also described.

A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.

Purification and characterization of a chimeric bifunctional antibody specific for human carcinoembryonic antigen and indium-benzyl-EDTA.

We describe the purification and characterization of a genetically engineered mouse/human chimeric bifunctional antibody specific for human carcinoembryonic antigen and indium-benzyl-EDTA. A clone expressing the bifunctional antibody has been previously described by our group and was found in this investigation also to express monospecific antibodies as well as Ig forms with mismatched light and heavy chains. The physicochemical properties of these various chimeric immunoglobulins were nearly identical. Isoelectric focusing showed that all these immunoglobulins have pI values between 8.47 and 8.80. A purification method that separates the bifunctional antibody from other Ig forms expressed in the same clone has been devised by relying on a unique interaction between the metal chelate binding region of these antibodies and the sulfopropyl functional group of a TSK SP 5-PW column.

Expression and characterization of a chimeric bifunctional antibody with therapeutic applications.

A simple method is described for the generation of a biologically produced mouse/human chimeric hetero-bifunctional antibody that has dual specificity for human carcinoembryonic Ag and metal chelate haptens. Two large compound chimeric vectors each containing the genetic information to produce a single antibody specificity were sequentially electroporated into the murine nonsecreting hybridoma SP2/0. This led to the isolation of a clone expressing high levels of total IgG (up to 25 micrograms/ml/10(6) cells), 10 to 20% of which showed simultaneous reactivity with both Ag. Binding studies showed that the immunoreactivities and affinity constants for the individual arms of the bifunctional antibody were equivalent to those seen with the parental antibodies.

Determination of the relative positions of amino acids by partial specific cleavages of end-labeled proteins.

We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.

Sequence homologies among E. coli ribosomal proteins: evidence for evolutionarily related groupings and internal duplications.

The complete or partial sequences of 47 E. coli ribosomal proteins described in the literature have been examined by computerized search and matching programs. In contrast to results previously reported by other investigators, sequence homologies were uncovered among some of these ribosomal proteins that are well beyond statistical expectations. Moreover, alignments of the most strongly homologous sequences suggested the existence of a network of family groupings. Several of these proteins also exhibit internal homologies, indicating that they have been elongated by a series of tandem duplications.