^36

Sydney Mangham.

ON SOME CRITICISMS OF THE OSAZONE

METHOD

OF DETECTING SUGARS IN PLANT TISSUES.

BY SYDNEY MANGHAM,

M.A.,

Lecturer in Botany, Armstrong College, Newcastle-on-Tyne,

in the University of Durham.^
INCE the publication of two papers^ dealing with the use of
phenylhydrazine for locating sugars in plant tissues some
criticisms of certain points dealt with have appeared, and have
been quoted. As these criticisms seem to be based in part upon
a somewhat inadequate appreciation of the significance of the
observations recorded, I propose to attempt to emphasise here
some points wliich I had hoped would have been clear to readers
of the papers in question.
In the interesting account by Davis, Daish and Sawyer^ of
their work on the carbohydrates of the mangold leaf a note
appears^ from which the following is extracted:
" It is difficult to understand Mangham's view that it is possible
to discriminate between dextrose and laevulose by means of the
osazone test, seeing that both sugars (as well as mannose) yield
identically the same osazone: Mangham seems to regard the
osazones from dextrose and l2evulose as distinct substances.
"In the writers'opinion little reliance can be placed on a
micro-chemical osazone test as a means of identifying maltose
in plant tissues, owing to the presence of large quantities of
other sugars. Our quantitative analyses (some 500 in all) have in
no single instance disclosed the presence of even traces of maltose
in the leaves or conducting systems of plants. In work of this
kind micro-chemical tests as a means of distinguishing individual
sugars should be avoided and only quantitative methods adopted.
Otherwise contradictory and uncertain results are inevitable."
In their review of recent work on carbon assimilation Jorgensen and Stiles^ make reference to the above criticisms, and add:

' Bacteriologist (Temporary), Naval Medical School, Royal Naval College,

Greenwich.
2 Mangham, S., 1911. " O n the Detection of Maltose in the Tissues of
certain Angiosperms." NEW PHYT. 10, pp. 160-166.
Mangham, 1915 " Observations on the Osazone method of locating sugars
in Plant Tissues. Ann. of Bot., XXIX, pp. 369 391.
' Davis, W. A., Daish, A. J., and Sawyer, G.C., 1910. " Studies of the Formation and Translocation of Carbohydrates in Plants. 1. The Carbohydrates
of the Mangold Leaf." Journ. Agric. Sci.. VII, pp. 25.')-326.
> I.e., p. 311.
Jorgensen, I., and Stiles, W., 1917. "Carbon Assimilation," NEW
PHYT. Reprint, No. 10, p. 128.

On Some Criticisms of the Osazone Method.

237

" The fact that Mangham should claim to distinguish hetween

rf-glucose and rf-fructose in the plant by means of the osazone test,
when their phenyl osazones are of course identical, is not very reassuring as to the dejjree of reliability of his results."
It is assumed by the above writers that in the papers dealt
with, a chemical difference is considered to exist between the
osazone yielded by dextrose and that yielded by Isevulose. Surely
this is somewhat gratuitous I It really loolts as if sufficient attention could not have been given to that section of the later paper
headed " Effects of Glycerine," a section occupying some fourteen
pages. A few extracts may perhaps serve to make this clear.
"The use of glycerine in the reagent has several advantages.
On the other hand it has some effect upon the reaction
with sugars, a point which does not appear to have received
adequate attention at the hands of other botanical workers or
critics who have dealt with it."'
" . . . . it has been found that the glycerine tends to
hinder or prevent crystal formation to an extent which varies with
the different sugars." ^
" This effect appears to be more pronounced with maltose and
dextrose than with levulose."^
A number of expei iments were described in which the effect of
glycerine was observed upon the production of osazones fi'om
sugars present in various concentrations. In the case of 1%
mixtures the following result was recorded.*
"The osazone came down more rapidly with levulose than
with dextrose [this difference was observed by Senft], and in the
former consisted of sheaves of long, fine, acicular crystals which
contrasted strongly with the more spherical and somewhat feathery
clusters of smaller though acicular crystals given by dextrose."
The actual extent of the " claim " to distinguish between dextrose and levulose by the use of Senft's reagent may be gathered
from the footnote occurring on the same page :
" It may be remarked that these two types of crystal clusters
can hardly be regarded as altogether distinctive characters for
dextrose and levulose. In low concentrations the difference is less
pronounced and may quite disappear."
Similarly in the summary :
"Too much reliance should not be placed on the crystal
cluster form as a feature distinguishing dextrose from levulose."">
1 I.e., p . 3 7 J .

' 1-C-. P - 3 ' 2 .

^ I.e., p. 374.

" I.e., p. 376.

' I.e., p . 3 8 8 .

238

Sydney Manghatn.

The effect of glycerine on the re-crystallisation of osazones

prepared from the various sugars used was also studied, the
osazones being dissolved in the glycerine by heating. It was found
that while some irregularities occurred, yet ". . . . crystallisation
seems to be retarded by the viscosity of the glycerine. Naturally in
a viscous medium the rate of diffusion of particles to form crystals is
slower than in a medium such as water." '
In the section entitled " Reaction with Mixtures of Sugars,"
attention was directed to the work of Scherman and Williams
who, using various concentrations of sugars, found that levulose,
in aqueous solution, precipitated its phenyl osazone in about onethird of the time required by the same concentration of dextrose.'
The point of all this is that d-glucose and J-fructose with
phenylhydrazine yield osazones of the same chemical compositioni
but that this osazone is formed in the one case from an aldose and
in the other from a ketose; the results of the experiments of
Senft, Sherman and Williams, and myself indicate that the formation of the osazone occurs more rapidly from rf-fructose than from
^/-glucose in aqueous solutions, while in strong glycerine the
viscosity of the medium retards the precipitation in both cases,
and to some extent also affects the configuration of the resulting
crystal clusters of the osazone, as shown by various figures and
photogi'aphs in the papers under consideration. It is.then a question of the influence of viscosity upon molecular rearrangements,
diffusion and crystal foi-mation.
With regard to the use of Senft's reagent for identifying
maltose, difficulties undoubtedly exist, but here again they are due
mainly to the influence of the glycerine ; this greatly retards, or
may completely prevent the production of crystals from the familiar
syrupy condition of the osazone.
This effect of glycerine was studied both by producing the
osazone from maltose in the presence of excess of strong glycerine,
and by observing the re-ctystallisation of the osazone from
glycerine in which it had been heated.'
As the result of the experiments performed the following
opinions were expressed:
" Considerable caution must therefore be exercised in attempting to locate maltose in starch-forming plants by means of Senft's
reagent. The formation of drops of syrupy liquid within cells,
especially if in any quantity, and if in tissues examined after starch
dissolution is known to have occurred, in all probability denotes
' lc.,p.3S\.

' /.c, 385-6.

' /.f., pp. 378 and 381.

On Some Criticisms of the Osazone Method.

239

the presence of maltose, though other possibilities are not altogether

excluded.
" If actual crystals are formed the osazone can be identified
with less uncertainty, thotigh heie again it is necessary to bear in
mind alternative interpretations."'
In this connection the work of Daish^ on the occurrence of
maltase in plants is of interest. This enzyme was found to be
present atmost universally in starch-forming plants, and to he
abundant in the leaves. Maltose, however, was not foimd hy
Davis, Daish and Sawyer^ in the leaves or conducting systems of
plants, and its absence was considered to be due to ". . . . the
amount of maltase always being in relative excess in the cells
where the starch degradation acttially occuis, so that it is able to
deal instantly with the whole of the maltose formed from the
starch. The fact that maltose, unlike cane sugar, never occtn's in
the stalks or conducting vessels is probably due to the fact that
maltase is an intracellular enzyme and apparently acts in close
collahoration and in the immediate proximity of the ordinary diastase which first attacks the starch in the cells where this substance
is stored."*
There is considerable interest in the above suggestion, although
some time may yet elapse before the distribution of intracellular
enzymes within the cell can be determined with any degree of
reliability.
It can readily be understood that an excess of maltase within
cells containing starch undergoing hydrolysis might prevent any
accumulation of maltose. But it can scarcely be that maltose
is completely absent from the cells at all times, for it must come
into existence for the maltase to act upon it, and until it can be
proved that the maltose is produced in actual contact with the
maltase it is reasonable to suppose that this sugar persists in the
cell, at least for the short time required for it to travel to the
enzyme prior to hydrolysis.
The ordinary equilibrium relations obtaining in enzyme actions
would presumably make the complete destruction of maltose within
the cell unlikely while any dextrose remained in the same cell. It
is, however, quite conceivable that owing to the activity and
abundance of the enzyme, although the total turnover of maltose
I.e., p. 379.
2 Daish A. J , 1916. " The Distribution of Maltase in Plants." Biochem.
J o u r n , X, pp. 31-76.
I.e., pp. 311, 313, 353, 354, 357, 358 etc.
4 /.f., p. 358.

240

Sydney Mangham,

might be great, yet at any one moment this sugar might be present
only in such small amount as to escape detection even by very
careful extraction methods.
In the course of my own worknot primarily directed towards
the determination of sugar changes in leavesno evidence for the
presence of maltose in anything but very small total amounts has
been found. What has been regarded as in all probability maltose
phenylosazone has been seen as a rule in small numbers of isolated
parenchymatous cells of leaf veins, and in the sieve-tubes of the
veins, for the most part the finest veins.
I can well believe that such amounts could easily escape
detection by methods involving extraction of leaf pulp, but I feel
as the result of spending much time in observing many sections of
plant tissue after treatment with Senft's reagent, and in examining
controls with pure sugars, that often maltose really can be detected
in this way. I have, however, never been able to demonstrate its
occurrence in anything in the least like the amount in which
hexoses occur. Indeed, the scarcity of maltose was formerly the
cause of considerable surprise in view of the results described by
Brown and Morris.
It should be almost unnecessary to insist that my papers on
the osazone method dealt with the results of work by means of
which it was sought to ascertain the degree of reliability of Senft's
reagent for diagnostic purposes.
This work has shown that by the use of Senft's reagent alone
it is not possible to distinguish with certainty the individual sugars
of a mixture, still less to determine the proportions in which they
are present.
While the reagent fails to furnish an ideal microchemical
sugar test suitable both for qualitative and quantitative work, yet
its use in certain forms of research is of considerable value, provided that its limits are always kept in mind, and that so far as
conclusions with regard to individual sugars are concerned the
results yielded are regarded as indicating probabilities rather than
as affording demonstrations.
At the present stage of inquiry into the nature of the changes
undergone by carbohydrates in foliage leaves, etc, it would be as
well to try to get the most out of all available means of attacking
the problems met with, in the hope that eventually sufficient
evidence may be accumulated and collated to enable conclusions
to be drawn with less uncertainty than is as yet possible, in spite of
the vast amount of laborious work which has been carried out,