Research on complex bactericidal compounds in the hemolymph of silkworms

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Hemolymph from 3, 000 fifth-instar silkworms was pooled, frozen and stored at -80ﾟC. Escherichia coli B/SM, a spontaneously occurring mutant strain resistant to streptomycin, was used for the estimation of bactericidal activity.By affinity chromatography using crab shell chitin powder, the hemolymph afforded a path through fraction (fraction A : eluted with a neutral buffer solution) and a lysozyme-like fraction (eluted with 0.1N acetic acid). Both fractions lysed Micrococcus Lysodeikticus. The lysozyme-like fraction alone could kill E. coli.The lysozyme-like fraction was further fractionated by a gel-filtration chromatography (GFC). The major fractions which killed E. coli on combination with the fraction A exhibited the bactericidal activity without the fraction A. The active fractions were combined and fractionated by a cation exchange chromatography to yield three lysozyme-like components, two of which killed E. coli without cofactors. The bactericidal activity of the major component, which was eluted from the cation exchange chromatography earliest among the three lysozyme-like fractions, was remarkably enhanced by addition of the fraction A. An electrophoresis of the major bactericidal component showed a single band having more basicity than egg-white lysozyme.Among the fractions obtained by GFC of the fraction A, there were three types of bactericidal components : 1) killed E. coli without lysozyme-like factors, and the addition of the lysozyme-like fraction did not seriously affect the activity, 2) killed E. coli without lysozyme-like factors and the addition of the lysozyme-like fraction diminished the activity, and 3) killed E. coli by combination with the lysozyme-like factors and the addition of the lysozyme-like fraction enhanced the activity remarkably. These facts indicate the presence of very complex mechanisms of selfdefense in the normal, cell-free hemolymph of silkworms.