An iodine-iodide reagent containing saturated calcium chloride was used for ... of iodine/potassium iodide and hemicellulose in the presence of sodium or ...
Biotechnology Techniques Received as revised 7th
XYLAN
SPECTROSCOPY
Vol 4 No 1 November
35-38
(1990)
AND SPECTROPHOTOMETRY
J.D. Fontana, A.M. Barbosa, M. Gebara, J.B.C. Correa, F. Reicher,’ M. Blumel, J.G. Chociai, M.C.O. Hauly, and KG. Johnson’ Department of Biochemistry, Universidade Federal do Parana, (81.504), CURITIBA, Parana, Brazil, and *Division of Biological Sciences,National ResearchCouncil of Canada, OTTAWA, Canada KlA OR6 An iodine-iodide reagent containing saturated calcium chloride was used for quantitation of larchwood xylan in solution and for improved visualization of xylanase activity on solid media. This spectroscopic methodology is applicable to hemicellulosesfrom other sources and can differentiate hemicelluloses from other polysaccharides such as amylose, amylopectin, and xyloglucan.
ABSTRACT.
INTRODUCTION
A variety of reagents including Congo Red, Ttypan Blue, and Graham’s Iodine (Williams, 1983) have been used for the detection of endoqlanase activity on solid media. Typically, endoxylanasesor organisms producing these enzymes are placed on plates of solid media containing xylan. After suitable reaction times, the plates are flooded with free dye to reveal activity as zones of clearing against a colored background. When using Graham’s Iodine as an endoxylanase detection reagent, it was observed that a second flooding with 1 M sodium or calcium chloride resulted in more intense color development (Williams, 1983). The interaction of iodine/potassium iodide and hemicellulose in the presenceof sodium or calcium chloride provided a useful tool to gain more insight into the nature and properties of hemicellulosic materials. For example, iodine/potassium iodine in the the presenceof CaCl, has been used in the fractionation of linear and ramified corn cob hemicelluloses(Gaillard, 1961). Confirming this finding, a differentiated interaction with iodine was observedwith hemicellulosesfrom C. arabica leaves following alkaline extraction (Wenzel and Correa, 1977). Since iodine/potassium iodide reagent in the presenceof saturated CaCl, has been successfullyusedin the calorimetric estimation of glycogen and other cr-glucans(Krisman, 1962), this study was undertaken to assess its efficacy with hemicellulosesof diverse origin.
MATERIALS
AND METHODS
Polysaccharides: Larchwood xylan, oat speltsxylan (Sigma), hemicellulose“A” from M. scabrella stem (Fontana er al., 1987), crude xyloglucan from cottonwood stem, potato amylose and amylopectin, and chemically 0-acetylated xylan (Johnson et nl., 1987)were usedin chromogenic assays.
35
Enzymes and Enzyme Assays: The hydrolase complex from the gastric juice of the snail M. paranaguensis (Fontana et al., 1987)and crude endoxylanasesfrom Streptomyces olivochrontogenes (NRCC 2258) were usedas xylan-depolymerizing enzymes. Enzyme reaction mixtures containing
0.5% (w/v) xylan, and 1 mg/mL protein in a final concentration of 10 mM sodium acetate buffer, pH 5.0 were incubated at 50°C for designatedintervals. Iodine Reagent (CIR): One mL of iodine (2.50 % w/v)-potassium iodide (25.0% w/v) diluted to 250 mL with saturated calcium chloride. Chromogenic
Chromogenic Assays: After the addition of 0.5 mL aliquots of xylan (50 to 500 pg/assay)freshly prepared in 10 mM sodium acetate buffer, pH 5.0 to 2.5 mL of CIR or Graham’s Iodine as indicated, absorption spectra of assayswere determined from 450 to 850 nm. RESULTS
The absorption spectra of unreacted CIR (Curve A) and Graham’s Iodine (Curve B) are presented in Figure 1. Significantly lessabsorption in the mid-visible light range was observed with CIR than with Graham’s Iodine. Addition of 500 pg larchwood xylan to CIR resulted in a dark blue color and the absorption spectrum presented in Curve A’ . Addition of the same amount of xylan to Graham’s Iodine, however, resulted in an absorption spectrum which was not essentiallydifferent from unreacted reagent (Curve B’ ). The large difference in absorption spectra of CIR before and after reaction with larchwood xylan therefore provides a potentially useful tool to study xylans in solution. Moreover, a linear relationship between concentrations of xylan up to 400 pg/assayand absorbanceat 600 nm was demonstrablewhen CIR was reacted with larchwood xylan. As indicated in Figure 2, the use of CIR also permitted spectroscopicdifferentiation of o(4-0methyl)-D-glucuronoxylans from distinct sourcessuch as oat spelts (Curve 7; absorption maximum, 590 nm), larchwood .(Curve
2; absorption maximum, 600 nm), and the leguminous
tree M. scabrelfa (Curve 1; absorption maximum, 610 nm). As well, this calorimetric technique allowed generation of spectrawhich distinguishedthe above acidic xylans from other iodophilic glycans such as xyloglucan (Curve 3; absorption maximum, 540 nm),
amylose (Curve 5;
absorption maximum, 655 nm), and amylopectin (Curve 6, absorption maximum, 490 nm). These materials similarily showedincreasedabsorbancewith increasedconcentration. Chemical or enzymatic modification of xylans had a profound effect on their interaction with CIR. For example, larchwood xylan that had been subjected to chemical 0acetylation gave absorption spectra similar to unreacted CIR (Curve 4, Figure 2). Similar spectra indicative of significantly reduced or completely abolishedreactivity with iodine were obtained when larchwood xylan was digested with snail or streptomycete endoxylanases. Even two minutes incubation with these enzymes resulted in altered spectra while after 10 minutes incubation there was virtually no reaction with CIR. Finally, the CIR reagent can be used for a rapid qualitative “ring” te:: for soluble xylans. Careful pipetting of xylan solutions onto the top of CIR generatesa deep blue colour at the interface. After vigorous mixing, the samesolution is ready for spectrophotometric use.
36
450
--
65U
550
---
150
850
nm Figure 1. Absorption spectra of CIR (Curve A), CIR plus larchwood xylan (Curve A'), Graham's Iodine (Curve B), and Graham's Iodine plus larchwood xylan (Curve B').
1.4
1.0
450
550
650
750
850
nm spectra of CIR plus E. scabrella xylan Figure 2. Absorption -____(Curve l), larchwood xylan (CurJe 2), crude xyloglucan 0-acetylated larchwood xylan (Curve (Curve 3), chemically (Curve 6), and oat spelts (Curve 5), amylopectin 4) I amylose xylan (Curve 7).
37
DISCUSSION Application
of specific spectroscopy
and quantitative spectrophotometry
is hampered to some degree by their characteristic
insolubility.
to hemicelluloses
The simplest procedure for
xylan isolation involves alkaline extraction followed by mild acidification
to pH 5.0 to recover
the main
1926).
fraction
iodine/potassium
as insolubilized
iodide-saturated
solubility but Lambert-Beer’s
hemicellulose
“A”
(O’Dwyer,
Use
of
the
CaCl, reagent described above does require some xylan
laws are satisfactorily
fulfilled for larchwood xylan up to 800 mg/L.
While alkaline pH is known to favour hemicellulose solubility, it is incompatible with the use of iodine as a chromogen. Routinely, xylan samples should be prepared in low ionic strength acidic buffers, and after xylan solutions should be recorded precipitation
are vigorously
mixed with CIR, absorption
as soon as possible since some P-glycan
of the polysaccharide-iodine
complex (Gaillard,
preparations
from
can lead to
1961).
Possibly the most potentially useful appIication of the interaction of heteroxylans
spectra
diverse
of CIR with x$ans is the
spectroscopic
differentiation
sources
hemicellulosic
CY-and P-glycans. Of particular interest is the behavior of 0-acetylated
xylans.
Since the hemicellulose backbone of native xylans frequently bears 0-acetyl substituents
(Biely,
1985), this feature deserves attention when spectrophotometric
and of other
non-
estimations are applied.
For
instance, careful isolation of the 44. scabrella stem heteroxylan preserving 0-acetyl content and position (Reicher et al., 1984) generatesa polysaccharidelacking reactivity with iodine. In addition to its use in spectrophotometric studiesof xylans, this iodometric reagent can also be used in more sophisticated applications such as detection of endoxylanaseactivity on agar or gellam gum electrophoretograms. ACKNOWLEDGEMENT: The financial support from CNPq, PADCT/FINEP and the Division of Biological Sciencesis acknowledged. REFERENCES Biely, P. (1985).
Trends in Biotechnology
3, 286-290.
Fontana, J.D., Gebara, M., Blumel, M., Schneider, H., MacKenzie, C.R., and Johnson, K.G. (1988). Methods in Enqwzology 160: 560-571. Gaillard, B.D.E. (1961). Nature
191, 1295-1296.
Johnson, K.G., Fontana, J.D., and MacKenzie, C.R. (1988). Methods 560. O’Dwyer, M.H. (1926). Biochent.
in Enqmology
160: 551-
J. 20, 656-664.
Reicher, F., Correa, J.B.C., and Gorin, J&P.
(1984). Carbohydr.
Wenzel, G.E., and Correa, J.B.C. (1977). An. Acad
Brasil.
Williams, AG., FEMS Microb. Lett. 20, 253-258 (1983).
38
Cienc.
Res. 135, 129-140.
49, 605613.
