Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

Abstract

The host-specific toxin of Helminthosporium carbonum (C32HsoNoO,o) was hydrolyzed by 6 N HCI to yield a number of a-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1 :2. Two other unstable a-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. (4,5,8,9,16,17 The most recent studies have been concerned with the hostspecific toxin of Helminthosporium carbonum Ullstrup, which is toxic to susceptible inbred dent corn (Zea inays L.) (3,6,7,14,15,19).Although each of these host-specific toxins has been isolated and crystallized in milligram quantities, because of the difficulty of isolation and their great instability only HC2 toxin has been characterized to any extent (5, 6, 7). It was shown to have a molecular formula approximating C32H.NMO,O by analysis of crystalline material and by analysis of a crystalline hydrochloride derivative. The molecular weight was determined by bioassay of the eluate of a Sephadex G-10 chromatography column and shown to be slightly less than 700. The toxin appeared to be a cyclic peptide, because, although it did not react with ninhydrin or fluorodinitrobenzene, after acid hydrolysis it yielded compounds which reacted to these reagents. The ultraviolet absorption spectrum of freshly lyophilized toxin dissolved in freshly distilled ethanol showed a single, well defined maximum at 230 nm. The molar absorptivity of a 1.4 X 10' M solution (assuming a molecular weight of 679) was approximately 5000. The position of the maximum and its relatively high intensity suggested a double bond in the vicinity of a carbonyl group, possibly an a , fl-unsaturated amide. Because the presence of such a function in HC toxin would explain the relative instability of the molecule, further work was done in characterizing the ninhydrin-reacting products of acid hydrolysis. MATERIAL AND METHODS

Phytotoxins Produced by Plant Parasites

“…Pringle & Scheffer (9 \) isolated that host-specifi c toxin of H car bonum, and Pringle (86) described it as being a cyclic peptide containing proline and alanine in a ratio of 1:2 with two unstable amino acids, one of which was identified as a-amino-2,3-dehydro-3-methylpentanoic acid. Since the molecular weight of the toxin was estimated to be about 700, it is conceivable that proline and alanine occur as one and two residues per mole, respectively.…”

Abolishment of Specific Toxicity of Host-Specific Toxin of Helminthosporium carbonum by Electrolytic Reduction

“…It is an unstable, cyclic, peptide-like compound. Acid hydrolysis of the toxin produces the amino acids alanine and proline in the molar ratio 2:1 and two additional ninhydrin-reacting products, one of which is 2-amino-2, 3-dehydro-3-methylpentanoic acid (a,/8-dehydroisoleucine) (7,8).…”

“…readily to form a sulfhydryl compound. An a, 8-unsaturated amino acid residue was found to be responsible for this reactivity and identified as 2-amino-2, 3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine (8). The addition of two hydrogen atoms by this procedure does not have a great effect on the physical properties of the molecule, but does render it much less reactive chemically because it has lost the reactivity characteristic of an a,,8-unsaturated amide (1).…”

“…A solution of 107.24 mg, dissolved in 10 ml of 10% aqueous isopropanol containing one drop of concentrated HCI, was divided into two parts. One part was reduced electrolytically in the cathode compartment of an electrolytic desalter, as described previously (8). After reduction was complete (10 min at room temperature), serial dilutions of both fractions, made with White's salts solution, were tested on germinated seedlings of susceptible and resistant corn, using the procedure described previously (15).…”

Chemical Constitution of the Host-Specific Toxin of Helminthosporium carbonum

“…A.F. Hill) (8,10,11). The most recent studies have been concerned with the host-specific toxin of Helminthosporium carbonum Ullstrup, which is toxic to susceptible inbred dent corn (Zea mays L.) (12,13,17).…”