A new simple and rapid chromatographic method for the purification of laccase and stellacyanin from Rhus vernicifera

.IRCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

123,63%646

(1968)

Communications A New Method

Simple

and

for

Rapid

the

Chromatographic

Purification

and Stellacyanin

from

of

Lactase

Rhus vernicifera’

Lactase and stellacyanin (1)) Rhus uernici~era blue protein (2), have been purified from Rhtcs eernicifern, a Japanese lac tree. The new simple method reported here includes chromatography on

The acetone powder (76 gm), which was derived from 1 kg latex, was stirred for 3 hours with 1500 ml of 0.05 ht sodium phosphate buffer, pH 5.5. The mixture was centrifuged at 2000g for 30 minutes. A powder of dry JIEAE-Sephadex (DEAE A-50, 3 gm) was mixed with the collected supernatant fraction and stirred for 30 minutes. The mixtuT was then applied on a T>EAE-Sephadex colur~~n (5 X 10 cm) equilibrated with the same buffer. A + 0.2M NaCl

4 I

=

. .._.. .&.

-

_._. . ---T!e-

FRACTION

Cmi”.)

FIG. 1. Elution pattern obtained on CM-Sephsdex chromatography. The deep blue but crude solut,ion of lacrase and stellacyanin (ultrafiltrate) was applied to the column (2.5 X 15 cm) equilibrated with 0.05 M sodium phosphate buffer, pH 5.5. After being washed with the same buffer, the column was eluted with the buffer containing 0.15 M SaCl, and fractions were collected every 3-10 minutes. The flow rate was 0.7 ml/minute. The lactase activity., which is shown as a broken line in t.he figure, was measured spectrophotometrically at ,j,j2 mp by using 5 mnc .V,-\T-dimethyl-p-phenylenediamine a,s the srthstrate at (&?/mintlte/lO ~1). (0), pII .i..j, Z”, in 0.0.3 M phosphate buffer containin, u 10 M M EDTA Absorbance at 610 rnp; (o), lactase activity.

two different types of ion-exchange Sephadex (Pharmacia, Uppsala, Sweden) and an ultrafiltration, and excludes the salting-out, procedures by ammonium sulfate and prolonged dialyses. The whole procedure described in this report was performed in the cold at 4-3”. 1 This work was sllpport ed in part, by a research grant, from Sorwegian Research Council of Science and IInmanitzies.

clear blue solution composed of a mixlnre of lnccase and stellacyanin was elrlted from the column. The brown and yellowish materials presenL in the ext,ract were almost complelely eliminated by this procedure. The solllt,ion was concen:rlltl .iO ml of deep bllle trated by ult rafiltratiolr, solrltion was obtailted. A part of this concentrated solut,ion (20 ml) was applied to a colrlmn of CM-Sephadex (CM C50) (2.5 X 15 cm) equilibrated with the buffer 638

1

B(St’?kyanin)

A(LaccaSe)

kO,/AzBO

:

= 0 158

604

1 $J--./(\

t 300

500

700 WAVE

300

450 500

700

1

LENGTH

FIG. 2. The nbsorptiou spectrum of the frwtions tlcsginnted as -4 nlltl 11 in Fig. 1. -1 pcr~rsnl of the resrdts indirat,es that the spert ra -I and II correspo~~l to those of laccnse nrrtl alld illdirates :t (.LP:LI.separation of the two components. The stcllacyanin, respectively, al~sorbancc ratio, 0.061 for lawase alld 0.138 for srellacyanin, agrees with the vnhles previor~sly reported, mid st,rongly suggests :I high ptlrity of t,he prepnrat ioIls.