A new serine protease from the latex of
Ipomoea carnea spp
. fistulosa (Morning glory), belonging tothe Con
vol
vulaceae family, was purified to homogeneity by ammonium sulfate fractionation followedby cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. ThepH and temperature optima for proteolytic acti
vity were 6.5 and 65
C, respecti
vely. The extinctioncoefficient (
2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35tryptophan, 76 tyrosine, and se
ven cysteine residues. The effect of se
veral inhibitors such as iodoaceticacid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsininhibitor, HgCl
2, 3
S-3-(
N-{(
S)-1-[
N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid,
N-ethyl maleimide, ethylene glycol-bis(
-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and
o-phenonthroline indicates that carnein belongs to the family ofserine proteases. The enzyme is not prone to autolysis e
ven at
very low concentrations. The N-terminalsequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarityto those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilasefamily endopepetidases.