Gallotannin biosynthesis
220
ated plastic bags[ Under these conditions\ the plant Leaves of staghorn sumac "79 g# were frozen in liquid
material could be stored for more than 5 months with! N1 and ground in a pre!cooled ultracentrifugal mill
out apparent loss of enzyme activity[
"Retsch KG\ Haan\ Germany#[ The frozen powder
Polyacrylamide!`el electrophoresis[ Anodic dis! was mixed with 79 g prewashed PVP and stirred for
continuous PAGE with 5) separating gels "pH 7[7# 29 min with a mixture of 014 ml Tris!HCl bu}er "0
and 3) stacking gels "pH 5[7# was employed for ana! M\ pH 7# and 014 ml borate bu}er "9[1 M\ pH 6[4#[
lysing the puri_cation of the enzyme ð04Ł^ to achieve The homogenate was squeezed through four layers
optimal separation\ all samples\ gels and bu}ers were of muslin\ followed by centrifugation of the _ltrate
supplemented with 9[0) Tween 19 ð05Ł[ In the case of "24\999 `\ 19 min#[ The supernatant crude extract was
denaturing PAGE "01[4) separating gel#\ this non! stirred for 04 min with 7 g Dowex 0X3 "49Ð099 mesh\
ionic detergent was replaced by 9[0) SDS[ Protein borate form# and _ltered through glass!wool[ A 1)
bands were detected by silver staining ð06Ł[
soln of protamine sulphate was added dropwise under
Enzyme activities were measured in standard assay stirring to the _ltrate until a _nal concn of 9[1 mg per
mixtures "69 ml vol[# containing 14 mM citrate bu}er 0 mg protein was reached^ after stirring for 29 min\
"pH 3#\ 149 nmol b!glucogallin\ 14 nmol pen! the solution was centrifuged "19\999 `\ 19 min#[ The
tagalloylglucose and 49 ml enzyme "for blanks\ heat! supernatant was fractionated with solid "NH3#1SO3^
denatured enzyme was used and b!glucogallin was the 24Ð69) ppt[ was redissolved in a minimal vol[ of
omitted#[ After incubation at 29> for 1 h\ the reaction 49 mM Tris!HCl\ pH 6[4 and subjected to gel!_l!
was stopped by adding 09 ml 0 N HCl[ Denatured tration on Sephacryl SÐ299 "column 39×1[3 cm i[d[#
protein was removed by centrifugation and the clear equilibrated in 49 mM Tris!HCl\ pH 6[4\ plus 9[0 M
supernatant lyophilised in a spin!freeze dryer[ The NaCl[ The most active frs "cf Fig[ 0# were concd by
dry residue was redissolved in 49 ml abs[ EtOH and ultra_ltration "Filtron Macrosep\ 09\999 MW
aliquots "4 ml# of this solution were analysed by nor! exclusion limit# and rechromatographed under ident!
mal!phase HPLC on LiChrosorb Si 59 "Merck CGC ical conditions[ The combined active frs were
glass cartridges^ particle size 4 mm^ column 049×2 adsorbed on DEAE cellulose "column 5[4×0 cm i[d[#
mm i[d[^ ~ow rate 0 ml min−0^ detection UV 179 nm#[ in 49 mM Tris!HCl[ pH 6[4\ plus 9[0 M NaCl[ After
For samples with tetra! to decagalloylglucoses a sol! washing out unbound protein\ the column was
vent composed of n!hexane!MeOH!THF!formic acid developed with a linear gradient of 9[0Ð9[4 M NaCl
"45]22]00]0\ supplemented with 399 mg oxalic acid which eluted the transferase at ca 9[3 M NaCl[ The
per liter ð07Ł# was used\ while assays with mono! to most active frs were adjusted to pH 4\ supplemented
tetragalloylglucoses were analysed with a less polar with "NH3#1SO3 "_nal concn 0[14 M# and adsorbed on
modi_cation of this solvent "52]16]8]0\ plus 219 mg butyl!Sepharose "column 0×0[7 cm i[d[# equilibrated
oxalic acid per liter ð01Ł#[ This comparatively fast ana! in 49 mM K!Pi bu}er\ pH 4\ plus 0[14 M "NH3#1SO3[
lytical method which\ however\ allowed only the sep! The column was washed with 0[14 M and 9[514 M
aration of galloylglucoses according to their sub! "NH3#1SO3 in bu}er\ followed by elution of the
stitution degree was used for the routine measurement enzyme with "NH3#1SO3!free bu}er[
of enzyme activities[ For the detailed determination
of individual galloylglucose isomers\ reversed!phase
Acknowled`ements*We thank Professor E[ Haslam
HPLC on LiChrospher 099 RP!07 "Merck LiCh!
"She.eld# and Dr H[ Schick "Heidelberg# for pro!
roCart cartridges^ particle size 4 mm^ column 149×3
viding rare galloylglucoses\ and Mrs A[ Muller for
Ã
mm i[d[^ detection UV 179 nm# with linear 9[94) aq[
H2PO3 "solvent A#:MeCN "solvent B# gradients was
employed[ Mono! to tetragalloylglucoses were sep!
arated with the following gradient] 9Ð1 min] 2) B\ 1Ð
3 min] 2Ð03)\ 3Ð19 min] 03Ð12) "~ow rate 0 ml
min−0#^ for penta! to heptagalloylglucoses the gradi!
ent was 9Ð1 min] 4) B\ 1Ð3 min] 4Ð07)\ 3Ð5 min]
07Ð10)\ 5Ð15 min] 10Ð14) "~ow rate 0[4 ml min−0#[
Quanti_cation of reaction products was done with
a computing integrator "Merck!Hitachi D!1499# and
referenced to ext[ standards[ A gallotannin degrading
esterase "{{tannase|| ð4Ł# in column eluates was deter!
mined by spot tests with the chromogenic substrate
naphthyl acetate ð08Ł[
Protein was determined colorimetrically according
to Bradford ð19Ł\ using BSA as standard[ Very dilute
solns were measured by UV photometry ð10Ł[
Enzyme puri_cation[ Unless otherwise stated\ all
operations were carried out at 9Ð3> and all bu}ers
were supplemented with 4 mM 1!mercaptoethanol[
excellent technical assistance[ Financial support by
the Deutsche Forschungsgemeinschaft "Bonn# and the
Fonds der Chemischen Industrie "Frankfurt:M[# is
gratefully acknowledged[
REFERENCES
0[ Gross\ G[ G[\ in Phenolic Metabolism in Plants\
ed[ H[ A[ Sta}ord and R[ K[ Ibrahim[ Recent
Advances in Phytochemistry\ 0881\ 15\ 186[
1[ Gross\ G[ G[\ in Plant Polyphenols] Synthesis\
Properties\ Si`ni_cance\ ed[ R[ W[ Hemingway
and P[ E[ Laks[ Plenum Press\ New York\ 0881\
p[ 32[
2[ Gross\ G[ G[\ in Comprehensive Natural Products
Chemistry[ Vol[ 2[ Carbohydrates and Their
Derivatives Includin` Tannins\ Cellulose\ and the
Related Li`nins\ ed[ B[ M[ Pinto[ Elsevier\ Oxford\
0887\ in press[
Niemetz, Ruth
