As biologically active substances, various kinds of proteins have been discovered and utilized for pharmaceuticals. It is very difficult to obtain enough amount of the proteins from natural source. Therefore, cell culture and DNA recombinant technology has been developed and made it possible to supply large amount of the proteins. The purification of proteins mainly consists of three steps, initial treatment, concentration or fractionation, and column chromatography. New and efficient separating materials for affinity chromatography and HPLC have considerably improved the isolation of proteins on the small and large scales. The amount of impurities, the most important being endotoxin, nucleic acids, and host cell proteins, are usually in the picogram range, and thus at the limit of sensitivity of the currently available methods.
Reagents and techniques for the analysis of primary structures of proteins have been reviewed with special emphasis on those which had been developed by the present author and his coworkers : Fluorescent Koshland reagent (FKR, 2-carboxy-1-hydroxy-4-naphtylmethyldimethylsulfonium chloride) for tryptophan residues and fluorescent Edman reagent (4-N, N-dimethylainino-l-naphthyl isothiocyanate, DNTC). FKR reacts with tryptophan residues at pH 03 and room temperature to produce fluorescent adduct (λex255 nm, λem 416 nm), the reaction being completed within 2 min. Owing to its high specificity, FKR was applicable to the determination of tryptophan residues in peptides and proteins. Methods for the selective isolation of tryptophan-containing fragments in peptides and proteins, based upon chelating ability of FKR derivative and reversed-phase high performance liquid chromatography, are described. Scope and limitation of DNTC in microsequence analysis of proteins are discussed, together with other Edman-type reagents. Furthermore, the reagents for protein assay and amino acid analysis are briefly reviewed.
Developments of high field nuclear magnetic resonance spectroscopy make us possible to determine the three dimensional structure of peptides and small proteins in solution. The procedure consists of three steps. (i) Peak assignments of every proton resonances to individual protons in a protein by means of several types of two dimensional NMR and isotope replacements. (ii) Measurements of NOE and translation to interatomic distances. (iii) Construction of molecular model which satisfies the NOE data by using distance geometry algorithm. Examples of the application of this method to a toxin peptide α-hANP will be shown, and limitations of the method and challenges to overcome them will be discussed.
The study of the structure of biofunctional proteins and receptors has become one of the most exciting and rapidly moving areas of life sciences. These fields are undergoing a rapid advancement with a tremendous growth of studies at the molecular level. The molecular resolution of the receptor protein reguires working with chemical methodologies and techniques. Photoaffinity labeling is a promising chemical approach for investigation of the structure of ligand binding sites within receptor proteins. The properties of various photogenerated reactive intermediates and its precursors are evaluated, while application of properly designed photolabeling reagents are discussed to highlight the potential of the method.
In this review I first described the stability of proteins obtained from unfolding experiments and how the replacement of a specified amino acid residue with other residues in a protein affects the protein stability. Second, I described the kinetics of unfolding and refolding of proteins. The transition from folded state to unfolded state or from unfolded state to folded state is highly cooperative, and no structural intermediate is detected. Therefore, it is difficult to understand how the regular structure of the native protein is formed from the unfolded molecule. Finally I described the pathway of the disulfide bond formation from reduced proteins.
Study of the sugar chains of various glycoproteins is now becoming one of the popular fields of biochemistry. This is because the finding of the functional roles of sugar moieties called the interest of researchers in many biological fields. Together with the physicochemical effects of the sugar chains of glycoproteins, roles of the sugar chains in signaling various cellular recognition phenomena have been reported. Development of recombinant DNA technique is also accelerating the research of sugar chains of glycoproteins, because many products did not express the expected biological activity. This review gives a summary account of our current knowledge of the sugar chain structures. Then, functional aspect of the sugar moieties of glycoproteins will be introduced with use of some representative cases.
Rapid advances in the fields of peptide chemistry and gene technology have resulted in an explosive accumulation of structural information on not only regulatory peptides but also their biosynthetic precursors and functional proteins such as enzyme, receptor protein, oncogene product and virus protein. For understanding the physiological systems in which the peptides and proteins are involved, well-characterized and purposely designed synthetic peptides are the most important and indispensable substrates. Synthetic replicates of regulatory peptides and their analogs and synthetic fragments of functional proteins have been prepared by chemical synthesis according to either classic or solid phase techniques. In this chapter, recent advances in the fields of peptide synthesis are described.
In order to clarify the structure-activity relationship of c-Ha-ras proteins (p 21 s) genes encoding p 21 (Val-12), p 21 (Leu-61) and p 21 (Arg-61) were synthesized by joining oligonucleotides with T 4 DNA ligase and expressed in E. coli under the regulation of E. coli tryptophan promoter. In addition, the gene for normal p 21 was constructed by cassette mutagenesis using restriction sites, ClaI-BssH II. The guanosine diphosphate (triphosphate) binding properties and guanosine triphosphatase (GTPase) activities of these p 21 s were examined. It was found that the guanine nucleotide binding abilities of all p 21 s produced in this study were relatively same but GTPase activity of activated p 21 s were significantly reduced. Furthermore, the gene encoding novel p 21 in which the guanine nucleotide binding sites was modified was synthesized by ligation of oligonucleotides and expressed in E.coli. Its biochemical activities are also discussed.
The molecular mechanism in the recognition of L-isoleucine and analog amino acids, by isoleucyl-tRNA synthetase, was elucidated by transferred nuclear Overhauser effect analysis of the conformations of amino acids bound to the enzyme. A variety of nonprotein amino acids, including furanomycin, were found to be incorporated into proteins in in vitro system. Furthermore, L-2-aminohexanoic acid (Ahx) was incorporated into human epidermal growth factor (hEGF), by using Escherichia coli strain harboring plasmid pTA 1522. From the successful production of Ahx-substituted hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.
Increasing efforts have been made for the development of antibody-directed, tumor-selective cytotoxic agents by coupling cytotoxic agents to antibodies to tumor-associated antigens on the tumor cell surface. Toxins and their subunits, anti-cancer drugs, and radioisotopes are used as the toxic agents for the antibody conjugates. This article discusses the current status of research on antibody conjugates with the enzymatic subunit A-chain of the plant toxin ricin and with the anti-cancer drug methotrexate focusing on the development of the methods of preparation of the conjugates having better biological profile.