A murine monoclonal antibody directed against human T-cell growth factor (TCGF) from the JURKAT cell line was used for affinity column.purification of the factor. Bound TCGF was eluted nearly quantitatively at low pH, and the recovered factor appeared homogeneous by two-dimensional gel electrophoresis. The molecule is markedly hydrophobic, with a high content of leucine. A single NH2-terminal sequence of 36 residues was obtained by automated Edman degradation, further supporting the homogeneity of the material. Thus, significant quantities of purified TCGF have been prepared in a single step, making possible detailed analysis of its molecular structure and biological role.

Human interleukin 2 was separated into multiple molecular forms by selective immunoaffinity chromatography and chromatofocusing. For the most part, this heterogeneity was attributed to variations in glycosylation of the threonine residue in position 3 of the polypeptide chain. The various molecular forms of interleukin 2 had nearly identical specific activities in the in vitro proliferation assay, indicating that the glycosylation had no significant effect on this response. The entire primary sequence of interleukin 2, including the location of the intramolecular disulfide bridge, was determined by a combination of peptide mapping and protein sequencing. This information should aid in the determination of the active site(s) of the molecule.

ABSTRACT2-Diazo-3,3,3-trifluoropropionyl chloride has been synthesized from trifluorodiazoethane and phosgene. Its derivatives are acid stable, can be used to label enzymes, and undergo photolysis with substantially less rearrangement than do derivatives of other known diazoacyl reagents designed for photoaffinity labeling. In particular, the diazotrifluoropropionyl thioester of methyl N-acetylcysteine undergoes photolysis in methanol with about 40% insertion into the -OH bond of the solvent; by contrast, photolysis of other diazoacyl thioesters gives substantially quantitative Wolff rearrangement. The trifluoro compounds hold promise for the photoaffinity labeling of thiols. Photoaffinity labeling was initiated with the photolysis of diazoacetyl chymotrypsin, prepared from the enzyme and pnitrophenyl diazoacetate (1, 2). Subsequently, this and other photolabile reagents have been applied (3-14) to a wide variety of problems in biochemistry and biology. (For reviews, see refs. 15-18.) Diazoacetates and diazomalonates (6, 7), however, suffer from the disadvantages that they are not acid stable, and that photolysis leads in large measure to the products of the photochemical analog of the Wolff rearrangement (2, 19). Furthermore, attempts to use the diazoesters of thiols, either with glyceraldehyde-3-phosphate dehydrogenaset or model compounds (20), have led only to products of the photochemical Wolff rearrangement or to other products that are not informative from the point of view of photoaffinity labeling. In this paper, we report the synthesis and photolysis of diazotrifluoropropionyl derivatives that are not subject to these limitations.2-Diazo-3,3,3-trifluoropropionyl chloride is easily prepared, and is purified by vacuum distillation; p-nitrophenyl diazotrifluoropropionate is a crystalline solid that can be purified by sublimation. Various

E. coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1). When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2). Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E. coli DNA topoisomerase I. dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme. dT8 is the shortest oligo(dT) that can be cleaved. The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide. No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long. dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length.

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