Rapid protein identification using N-terminal "sequence tag" and amino acid analysis.

Proteins can be identified by amino acid analysis and database matching, but it is often desirable to increase the confidence in identity through the use of other techniques. Here we describe a rapid protein identification method that uses Edman degradation to create a 3 or 4 amino acid N-terminal "sequence tag," following which proteins are subjected to amino acid analysis protein identification procedures. Edman degradation methods have been modified to take only 23 min per cycle, and rapid amino acid analysis techniques are used. The Edman degradation and amino acid analysis is done on a single PVDF membrane-bound protein sample. A computer database matching program is also presented which uses both amino acid composition and "sequence tag" data for protein identification. This method represents the most inexpensive, accurate, and rapid means of protein identification, which is ideal for the screening of proteomes separated by 2-D gel electrophoresis. The creation of N-terminal Edman degradation "sequence-tags" prior to peptide mass fingerprinting of samples should also be useful.

Automated subpicomole protein sequencing using an alternative postcleavage conversion chemistry.

An automated subpicomole sequencing method is described. It is based on a chemistry that generates easily detectable amino acid derivatives from the postcleavage products of the classical Edman degradation. These products, which include the amino acid anilinothiazolinone (ATZ), phenylthiocarbamyl (PTC), and phenylthiohydantoin (PTH), are converted to a homogeneous preparation of ATZ that can react with sensitivity-enhancing compounds such as fluorescent amines. The method is demonstrated with a protein of known sequence (alpha-lactalbumin) and appears to offer a significant improvement over current high-sensitivity protein sequencing strategies. Modifications to the sequencer hardware or to the Edman degradation chemistry are not required.

The generation of phenylthiocarbamyl or anilinothiazolinone amino acids from the postcleavage products of the Edman degradation.

A method is described for the generation of phenylthiocarbamyl (PTC) amino acids from phenylthiohydrantoin (PTH) or anilinothiazolinone (ATZ) amino acids. An aqueous base containing a reducing agent promotes the opening of the PTH or ATZ ring to form a stable PTC amino acid. The new reaction is essentially quantitative for all of the amino acids and may be used instead of the established Edman conversion with an aqueous acid. The PTC amino acids may be analyzed directly or used for generating other analytes. A further method is described for the generation of ATZ amino acids from either the PTH or PTC derivatives. This chemistry involves a Lewis acid-catalyzed dehydration of the PTC. The resulting ATZ may be combined with labels that permit high-sensitivity detection. This reaction is also essentially quantitative for all of the amino acids except aspartic acid, which exhibits a strong preference for the PTH form under these conditions. Taken together, these chemistries allow the preparation of homogeneous PTC or ATZ amino acids from any mixture of amino acid derivatives resulting from the Edman degradation and promise to greatly facilitate the high-sensitivity analysis of protein.

Reducing chemical background noise in automated protein sequencers.

Baseline noise resulting from the by-products of the automated Edman degradation of proteins is reduced using nonstandard coupling chemicals and dual-wavelength diode array detection of phenylthiohydantoin (PTH) amino acids. Changes to the chemistry include lowering the phenylisothiocyanate (PITC) concentration and using a novel coupling base diisopropylethylamine (DIPEA). The diode array is configured to cancel the UV absorbance of reaction by-products without adversely affecting the PTH signal intensities. Combining these techniques gives a significant reduction in chemical noise on analytical chromatograms. Less sample is required for sequence analysis since low-level amino acid peaks are more easily identified.

Studies on C3ahu binding to human eosinophils: characterization of binding.

C3a purified to chemical homogeneity from human serum binds preferentially to human eosinophils greater than neutrophils. Little or no binding is found with human platelets. Maximum binding to eosinophils at 37 degrees C occurs within 15 min. Dilution of 125I-C3a by either cold C3a or washing away unbound 125I-C3a and reincubating at 37 degrees C reveals a T1/2 of approximately 30 min. C3adesArg neither binds to eosinophils nor inhibits the binding of 125I-C3a. The binding of C3a to human eosinophils may reflect a physiologic role of C3a in eosinophil motility or function.

Immunoglobulin allotypes of rabbit kappa chains: polymorphism of a control mechanism regulating closely linked duplicated genes?

The amino acid sequence of the constant (CK) region from the kappa immunoglobulin chains of a b9 rabbit is compared with the CK sequences, taken from the literature, of a b4 rabbit. These CK regions differ by 33% of their amino acid sequences and by three sequence insertions or deletions (sequence gaps). These extensive differences together with other published observations suggest that the b9 and b4 CK genes may not be simple alleles, but rather they may be encoded by closely linked CK genes present in every rabbit whose expression is regulated by a polymorphic control mechanism.

Mouse immunoglobulin heavy chains are coded by multiple germ line variable region genes.

The N-terminal 20 residues of 13 heavy immunoglobulin chains from myeloma protein of the BALB/c mouse are compared with the same residues of 15 other heavy chains described in the literature. Sixteen of 28 sequences are different from one another. These proteins fall into four major sets, with 18 of the proteins in the largest set being further divisible into at least five subsets. This pattern of diversity suggests there are at least eight germ line genes coding for the variable regions of mouse heavy chain. Many of the immunoglobulins from which these heavy chains are derived exhibit binding activity for various haptens. The differing hapten specificities are closely correlated with distinct primary amino-acid sequences.