Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.

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White face hornet (Dolichovespula maculata) venom has three known protein allergens which induce IgE response in susceptible people. They are antigen 5, phospholipase A1, and hyaluronidase, also known as Dol m 5, 1, and 2, respectively. We have cloned Dol m 2, a protein of 331 residues. When expressed in bacteria, a mixture of recombinant Dol m 2 and its fragments was obtained. The fragments were apparently generated by proteolysis of a Met-Met bond at residue 122, as they were not observed for a Dol m 2 mutant with a Leu-Met bond. Dol m 2 has 56% sequence identity with the honey bee venom allergen hyaluronidase and 27% identity with PH-20, a human sperm protein with hyaluronidase activity. A common feature of hornet venom allergens is their sequence identity with other proteins in our environment. We showed previously the sequence identity of Dol m 5 with a plant protein and a mammalian testis protein and of Dol m 1 with mammalian lipases. In BALB/c mice, Dol m 2 and bee hyaluronidase showed cross-reactivity at both antibody and T cell levels. These findings are relevant to some patients' multiple sensitivity to hornet and bee stings.

We have determined the sequence of a venom allergen phospholipase A~ from white-faced hornet (Dolichovespula maculata) by cDNA and protein sequencings. This protein of 300 amino acid residues (Dol m I) has no sequence similarity with other known phospholipases. But it has sequence similarity with mammalian lipases; about 40% identity in overlaps of 123 residues. Tests suggest that hornet phospholipase has weak lipase activity. Hornet venom has 3 major allergens, and another hornet allergen antigen 5 (Dol m V) was previously found to have sequence similarity with a mammalian testis protein and a plant leaf protein.Hornet venom allergen; Phospholipase A~

Chemically modified allergens may be useful immunotherapeutic reagents in man. What is required of such reagents is that the modified allergens show reduced allergenic activities yet retain the immunosuppressive properties of the native molecule. It is with this objective that we have prepared conjugates of antigen E with two polymers of large molecular weights. Antigen E is the major allergen of ragweed pollen and it is an acidic protein of ~38,000 daltons (1). The two polymers are methoxypolyethylene glycol (MPEG) 1 of ~5,000 daltons and a copolymer of Dglutamie acid and D-lysine (DGL) of ~34,000 daltons. MPEG was chosen because MPEG protein conjugates are known to have reduced allergenic and immunogenic activities (2, 3). DGL was chosen because DGL conjugates with haptens of low molecular weight are known to possess hapten-specific immunosuppressive properties (4, 5). To study the influence of the bulky MPEG groups on the immunochemical properties of the conjugate, we also prepared a conjugate using methanol in place of MPEG.In this paper we report comparative studies on the immunological properties of antigen E and its conjugates in the mouse as the experimental animal. The mouse system was chosen since it is generally accepted that the IgE response in this system is the best model for reaginic antibody formation in man (6). MPEG and methoxy antigen E conjugates were prepared by coupling the protein with cyanuric chloride activated MPEG (2) or methanol (7) as shown in Fig. 1. DGL antigen E conjugate was prepared via an intermolecular disulfide bond formation (8). As shown in Fig. 2, antigen E and DGL were first modified to contain reactive thiol and 4-dithiopyridyl groups, then they were coupled via these groups. Materials and Methods Materials.Antigen E was isolated from ragweed pollen as described (9); its isoelectric forms B and C were used interehangeably in this study. MPEG of average mol wt of 5,000 was a gift of Union Carbide Corp., New York. A random copolymer of DGL in a molar ratio of 6:4, having an average mol wt of 34,000, was a gift from Miles