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that has been stewed for about half an hour.

(p) A white precipitate is formed not given with peptones on
adding a little glacial acetic acid, then potassium ferrocyanide
solution drop by drop.

(q) Add excess of acetic acid, then an equal volume of saturated
sodium sulphate solution, and heat ; the precipitate formed removes
all proteins (except peptones) from a solution.

(r) Saturate the solution with ammonium sulphate, by adding
crystals or the powdered salt until no more will dissolve on shaking
a white precipitate, not given with peptones. This throws down
all proteins (except peptones) from solution ; filter, and note that
the filtrate now contains no proteins



CELL-CONTENTS AND CELL-WALLS. 4l

(s) Dry the clotted albumin of a hard-boiled egg, mix it with
about twice as much powdered soda-lime, and add a little water to
form a paste of the mixture. Roll this paste between the fingers
into small pellets, and place these in a dry warmed tube of hard
glass. Heat over a Bunsen, and into the mouth of the tube place
first (A) a moist red litmus paper, then (B) a lead acetate paper.
The escaping vapours turn A blue and B black ; the former change
is due to ammonia (which can also be smelt), the latter to the for-
mation of lead sulphide proving the presence of nitrogen and of
sulphur in the albumin.

(t) Put a bit of hard-boiled egg on a needle, and hold in a Bunsen
flame ; it becomes charred, showing that carbon is present.

42. Froteoses and Peptones. These derivatives of
proteins ( 40) are formed in nature by the action of pro-
teolytic enzymes (pepsin, trypsin) on the primary proteins.
It is doubtful whether they occur as reserve food in resting
seeds, but they appear when germination begins.

The proteoses (soluble in water, not coagulated on boil-
ing, but precipitated by acids) are intermediate digestion
products between primary proteins and the peptones
(soluble in water and neither coagulated by boiling nor
precipitated by acids). The peptones are readily soluble
in water, and are not precipitated by acids, alkalis, neutral
salts, and many of the other reagents that precipitate the
primary proteins. The proteoses are less diffusible than
the peptones ; some proteoses are not readily soluble in
water, and they are distinguished from peptones by being
precipitated when their solutions are saturated with ammo-
nium sulphate. Proteoses yield precipitates with many of
the reagents that precipitate other proteins ; the precipitates
they give with nitric acid, and with potassium ferrocyanide
in presence of acetic acid, disappear on warming and
reappear on cooling.

43. Experiments with Commercial Peptone. Get some
Witte Peptone, which in reality contains more proteose than true
peptone. Dissolve in warm water, and make the following tests for
proteose and for peptone, after dividing the solution into portions
in test-tubes.

(1) Heat the solution and acidify it with dilute acetic acid no
coagulation. (2) Saturate with ammonium sulphate a white pre-
cipitate, which partly disappears on heating and reappears on



42 CELL-CONTENTS AND CELL-WALLS.

cooling. (3) Add nitric acid a white precipitate, which dissolves
on heating, the liquid turning yellow, and reappears on cooling.
(4) The Biuret test a rose pink colour. (5) Add acetic acid and
potassium ferrocyanide, (6) saturate with common salt in each
case a precipitate, which disappears on heating and reappears on
cooling.

Now add ammonium sulphate to saturation to the remainder of
the solution, filter, and to the filtrate (which contains peptone but
not proteose) apply the general protein tests xanthoproteic,
Millon's, biuret (rose pink colour given) ; note also that the filtrate
gives no precipitate with acids, or with acetic acid and potassium
ferrocyanide.

44. Dialysis Experiments with Albumin and Pep-
tone. Fit up two dialysers (Fig. 20), each floating in a

dish of distilled water.
Into A place some of
the albumin solution,
into B some peptone
solution ; to each add
a little thymol or other
antiseptic. Let the two
dialysers stand for
three days ; then test

Fig. 20. A Dialyser, made by binding parch- " 1G water in 6aC J 1 J
ment paper over a hoop of rubber. proteins, USHlg ( With

different samples) the
xanthoproteic, Millon's, biuret, and other tests.

Note that albumin is indiffusible, while peptone is diffu-
sible, though somewhat slowly, through a membrane.

45. Proteins in Pea Flour. Pea flour contains starch,
dextrin, and several proteins. The chief protein is a globulin
(legumin), but there is also another globulin (vicilin) and an albu-
min (legumelin).

(a) Place 10 grams of Pea flour with 50 c.c. of water in a flask,
shake vigorously, let it stand for several hours, and filter. Test
the residue tor starch with iodine. Divide the clear filtrate, con-
taining the albumin, into several parts and apply to these the chief
protein tests : (1) Xanthoproteic test ; (2) Millon's test ; (3) Biuret
test ; (4) acetic acid and potassium ferrocyanide ; (5) heat and note
the coagulation of the albumin, especially if a few drops of acetic
acid be added.




CELL-CONTENTS AND CELL-WALLS. 43

(6) Treat some Pea flour with 10 per cent, salt solution for several
hours, and filter ; test the residue for starch. To portions of the
clear filtrate apply the chief protein tests ; then drop some of it
into a beaker of water note the precipitate of globulins.

46. Proteins in Potato Tuber. Scrape the surface of a potato
into a beaker ; to the scrapings add some salt solution, stir well,
and strain through calico into another beaker. On standing, a
deposit of starch is formed ; examine this with the microscope, and
test a portion of it with iodine. Pour off the liquid, and apply to
it the chief protein tests.

47. Proteins in Wheat Plour. Make extracts of ordinary
wheaten flour with (1) water, (2) salt solution, (3) alcohol. In each
case filter, and test the filtrate for proteins.

Separate the gluten (a mixture of proteins) from the starch, as
follows. Enclose a tablespoonful of flour in a piece of fine muslin,
and knead it in a basin of water. Note the deposit of starch
grains ; examine these with the microscope and compare with those
of Potato and Pea. Remove the starch entirely by kneading under
a running tap until the water at first whitened by the starch
passes off clear ; open the muslin and note the yellowish sticky
mass of gluten left behind.

Extract the gliadin from the gluten by boiling with alcohol,
filter, evaporate the alcohol from the filtrate, and apply the protein
tests to the residue (gliadin). The insoluble remainder left on the
filter contains glutelin ; note that this is insoluble in water and in
salt solutions, soluble in dilute acetic acid and in dilute caustic
soda.

Prove the presence of carbon, nitrogen, and sulphur in (1) Pea
flour, (2) the "gluten" just isolated from Wheaten flour in the
same way as with egg-albumin ( 41, s, t).

48. Proteins in Brazil Nut. Remove the shells from some
seeds, grind up the seeds, and extract with ether to remove the oil ;
this may be done best with a Soxhlet fat extraction apparatus
( 97). Allow the ether to eA^aporate, and note the residue of oil.

Extract about 10 grams of the oil-free nut meal with 50 c.c. of
10 per cent, salt solution. Pour some of the extract into about 20
times its volume of water in a beaker ; a cloudiness is produced
which on standing separates into flakes and falls to the bottom.
Then pour off the greater part of the water, and filter the remain-
der ; to the precipitate apply the chief protein tests.

The crystalline globulin (excelsin) of Brazil nut can be obtained
in fine hexagonal plates by dialysing the saline extract ; by this
method the globulin separates out more slowly than by simply
pouring the extract into water.



44 CELL-CONTENTS AND CELL-WALLS.

49. Microchemical Tests for Proteins. All the

tests that give a colour reaction may be readily used as
microchemical methods of detecting proteins in plant
tissues.

(a) Cut thin sections from a Pea, Bean, or Lupin,
cotyledon. (1) Treat a section with iodine ; the starch-
grains turn blue, the small protein grains turn brown or
yellow. (2) Lay a section in strong copper sulphate
solution for a minute, rinse in water, and transfer to a
little potash in a test-tube, heat to boiling ; mount, cover,
and note the violet colour of the protein cell-contents.
(3) Apply the xanthoproteic test, by placing a section
first in strong nitric acid in a watch-glass, then in strong
ammonia ; note the intense yellow colour of the protein
contents. (4) Place a section in a little Millon's reagent ;
if the protein contents do not turn red quickly, warm the
slide. See also 74.

(6) Cut transverse and longitudinal sections of the grain
( " seed " ) of Wheat and of Maize and apply the above
tests. Note that the greater portion of the endosperm
consists of cells packed with starch-grains, but the outer-
most layer (" aleurone " layer) consists of cubical cells
containing protein grains. See also 75.

50. Protein Grains ("Aleurone" Grains) are found
in various parts where food is stored, but are especially
abundant and large in seeds. In some cases the grains
are small and of simple structure ( 51). In other cases,
especially in oily seeds, they are large ( 52) and contain
one or more angular " crystalloids " (protein crystals) and
also rounded " globoids " consisting of mineral substance
(double phosphate of calcium and magnesium). Protein
crystals may also occur in cells quite apart from definite
protein grains.

51. Simple Protein Grains. Get dry seeds of Al-
mond, Apple, Bean, Pea, Lupiu, Sunflower. Moisten the
razor with glycerine, cut sections of the cotyledons, and
mount in glycerine.



CELL-CONTENTS AND CELL-WALLS. 45

Note the numerous small refractive protein grains,
which at first sight may resemble starch grains, but are
not stratified and turn brown, not blue, with iodine. Of
these simple grains, some are soluble in water (Almond,
Apple) ; others, insoluble in water, are soluble in saturated
salt solution either at once (Beans, Peas, Lupin), or after
treatment with alcohol (Sunflower).

In each case treat different sections with (1) water
even when this does not dissolve the grains, it usually
makes them swell and lose their bright appearance ;

(2) potash this makes the grains swell and dissolve ;

(3) iodine this turns the grains brown ; (4) Biuret test ;
(5) Xanthoproteic test ; (6) Millon's reagent.

52. Protein Grains with Crystalloids and Globoids.

Brazil nut and Castor Oil seeds form good material
for the study of the larger and more complex protein
or " aleuroue " grains, which are embedded in the oil-
containing protoplasmic matrix of the cells. These grains
are not soluble in water, but are dissolved by strong salt
solution, either at once (Brazil nut) or after treatment
with alcohol (Castor Oil, Walnut). In each case remove
the shell, and make the following preparations. In each
case cut the sections with the razor dry, except where
otherwise directed.

(a) Mount dry sections in thick glycerine the oily
matrix of the cells will be seen, with the oil drops ; note
the protein grains, in which the crystalloids and globoids
may be seen.

(b) Mount sections in olive oil (which may in this case
be used for wetting the razor). Note that the oil makes the
oily matrix of the cells transparent and almost invisible.

(c) Wet the razor with alcohol, cut sections, soak them
in alcohol to dissolve out the oil (ether will do this more
quickly wash out the ether with alcohol), and mount in
thick glycerine.

(d) Cut dry sections, and mount them in water this
makes the grains swell, but the crystalloids should become
more conspicuous. Irrigate sections, mounted in water,



46 CELL-CONTENTS AND CELL-WALLS.

with (1) iodine solution the grains turn yellow ; (2) dilute
potash the crystalloids swell and dissolve, leaving the
globoids behind ; (3) dilute sulphuric acid (note that this
destroys the grains), then iodine solution (this stains the
matrix left behind in the cells) ; (4) a drop or two of
1 per cent, osniic acid the crystalloids slowly swell, while
the rest of the cell- contents, especially the oily matrix,
rapidly becomes blackened.

(e) Cut dry sections, and transfer them to a watch-glass
containing two parts of alcohol and one part of castor oil,
with enough eosin to make the mixture light red. After
a few hours, mount in castor oil and alcohol (without the
eosin) . This treatment brings the grains out clearly ; they
are seen embedded in vacuoles in the cytoplasm of the cells.

(/) Place some dry sections in alkannin ( 86) for
several hours, and mount in dilute glycerine ; the oil is
stained red.

(g) The structure of the grains is well brought out by
fixation in alcoholic picric acid, and staining with eosin.
Place the sections in concentrated alcoholic solution of
picric acid in a watch-glass for several hours ; then wash
them in alcohol, and stain for a few minutes in eosin
dissolved in alcohol. It is best to wash the sections next
in absolute alcohol, transfer them to oil of cloves, and
mount in Canada balsam. The matrix of the grains is
stained dark red, the crystalloid yellow, and the globoid
remains colourless.

(h) Note that the globoids are (1) insoluble in alcohol
and in dilute potash, but (2) soluble in dilute mineral
acids (hydrochloric, nitric, or sulphuric) and in acetic
acid ; (3) in an ammoniacal solution of ammonium phos-
phate the globoids are replaced by crystals of ammonium
magnesium phosphate ; (4) on being treated with am-
monium oxalate, they are replaced by crystals of calcium
oxalate ; (5) after extracting the oil from sections by treat-
ment with alcohol, or alcohol and ether, the globoids can
be made to stand out clearly on adding some dilute ( 1 per
cent.) potash solution which will dissolve the ground sub-
stance of the protein grains,



CELL-CONTENTS AND CELL-WALLS. 47

(i) Place some sections in a- watch-glass containing
either pepsin or trypsin, e.g. liquor pepticus ( 54) or
liquor pancreaticus ( 57) ; for comparison, place others
in a watch-glass of water. Set both in a warm place, and
note that the ground substance of the protein grains is
first dissolved, then the crystalloid more slowly, while the
limiting membrane of the vacuole occupied by the grain
persists.

53. Digestion of Proteins. In mammals the pri-
mary proteins are acted upon by the gastric juice of the
stomach and by the pancreatic juice and the intestinal
juice (succus entericus) of the small intestine. The hy-
drolysis of the proteins is effected by the three enzymes,
pepsin, trypsin, and erepsin, present in these three juices
respectively. Pepsin hydrolyses the primary proteins into
peptones ; trypsin also acts upon the primary proteins, but
it carries the hydrolysing process further and changes the
peptones into amino-acids ; erepsin is peculiar in that it
does not attack the primary proteins, but is only capable
of acting upon proteoses and peptones, changing them into
amino-acids.

For our purposes we may regard the proteolytic enzymes
of plants as corresponding to trypsin in their mode of
action. The vegetable trypsin called papain is obtainable
commercially, being used in medicine, but for the follow-
ing experiments we may use either pepsin prepared from
gastric juice, or preparations of pancreas containing the
enzymes diastase and lipase in addition to trypsin.

54. Preparation of Pepsin. (a) Pepsin may be purchased in
the form of Beiiger's " liquor pepticus," or the dried pepsin (Bur-
roughs and Wellcome). (6) Artificial gastric juice may be prepared
as follows Get a fresh pig's stomach from the butcher, cut it open,
rinse with water, cut out the cardiac (broader) end, spread it out,
scrape the mucous (inner) surface, rub up the scrapings in a mortar
with sand, add water, rub up again, and filter ; the filtrate is to be
used. Another method is to scrape the mucous membrane off, dry
the scrapings between folds of blotting-paper, put them in a
bottle, and cover with glycerine which will dissolve out the pepsin ;
after a day, filter, and use the filtrate (glycerine extract),



48 CELL-CONTENTS AND CELL-WALLS.

55. Experiments with Pepsin. Boil an egg hard, and chop
the clotted white into small pieces. Label six test-tubes A, B, C,
.Z), E, F. Half fill each tube with water, and drop in some of the
chopped albumin. To A add some pepsin extract or some pepsin
powder, with a pinch of bicarbonate of soda to make the liquid dis-
tinctly alkaline ; to B and C add some pepsin and a few drops of
dilute hydrochloric acid ; to D add a few drops of acid, but no
pepsin ; to E, some acid together with pepsin extract (or dissolved
pepsin powder) which has been boiled ; and leave F with nothing
added to the albumin.

Set all the tubes, except C, in a beaker of warm water, and keep
at 40 C. on a bath for an hour. Put C in a freezing mixture, or ice
and water, for the same period. Note that in A, C, E, and ^ 7 tho
albumin is unchanged ; in B it has disappeared, having become
swollen up and clear.

Now apply to a few drops of liquid from each tube the xantho-
proteic and the biuret tests. Peptone is present in B, but not in
any of the others. In E the pepsin has been destroyed by the boil-
ing. In A the action of the pepsin has been prevented by the
alkaline medium ; on adding acid to the liquid and keeping the
tube at 40 C. again digestion takes place. In C the action has been
prevented by the cold ; on transferring the tube to the bath at
40 C. digestion takes place. In Z>, the weak acid used, without
pepsin, has only changed the albumin into acid-albumin, but not
into peptone.

56. Products of Peptic Digestion. Repeat the preceding
experiment on a larger scale, so as to get more material to test for
the products of pepsin action. This time place in a flask some
pieces of albumin, dilute hydrochloric acid (add 4 c.c. of strong
acid to 300 c.c. of water), and some pepsin extract or powder. Keep
at 40 C. for an hour ; if the liquid is cloudy, filter it.

(A) To the liquid, or nitrate, add dilute caustic soda solution
until it becomes neutral a precipitate is given, consisting of acid-
albumin ; filter off this precipitate, dissolve it in dilute acid, and
note that the acid solution gives protein reactions and does not
coagulate on boiling.

(B) Test part of the filtrate from A for proteose. It gives the
protein reactions. On adding nitric acid and common salt, a pre-
cipitate is formed, which is re-dissolved on heating but reappears
on cooling. It is precipitated by (a) acetic acid and potassium
ferrocyanide, and by (b) acetic acid and saturated sodium sulphate
solution, neither of which precipitates peptones. It gives the same
biuret reaction (rosy pink) as peptones and, like them, is soluble in
water.

(C) Saturate another portion of the filtrate from B with ammonium
sulphate crystals, or the powdered salt ; this precipitates the pro-
teoses, while the peptones remain in solution test with biuret,
using a large amount of soda.



CELL-CONTENTS AND CELL-WALLS. 49

57. Preparation of Trypsin. There are various commercial
preparations which contain trypsin, e.y. Benger's "liquor pancrea-
ticus " (which often contains a sediment of tyrosin), the " Holadin "
of Fairchild Bros, (a very active preparation containing also lipase
and diastase). The vegetable trypsin, papain, can also be obtained ;
it contains only trypsin.

To make a glycerine extract of pancreas, which will serve also
for experiments on the hydrolysis of starch ( 74) and that of oils
( 86), mince up a fresh ox or pig pancreas ("sweetbread") in the
same way as directed for the gastric extract ( 54).

58. Experiments with Trypsin. Repeat the experiments
directed for pepsin ( 55), but instead of acid use 1 per cent, sodium
bicarbonate solution. To prevent putrefaction, add some anti-
septic such as thymol, or toluene, or chloroform water (5 c.c. of
chloroform shaken with a litre of water).

Label thiee test-tubes A, B, C. Half fill each with 1 per cent,
sodium carbonate solution, and add some hard-boiled egg white,
with a few drops of the antiseptic. Boil B ; make G acid with
dilute hydrochloric acid. Plug the three tubes with cotton-wool,
and place them in a bath at 40 for an hour. In A the liquid
becomes more or less clear, the albumin being digested ; in B and
C there is no change.

Filter the liquid in A , neutralise the filtrate with dilute acid ;
alkali albumin is precipitated filter this precipitate off and test
the filtrate for peptones.

Filter B and C, and neutralise B with acid and C with sodium
carbonate ; no precipitate is formed. Test for peptones none are
present. In B the trypsin has been destroyed by the boiling, in
C its action is prevented by the presence of the acid.

59. Products of Tryptic Digestion. Make a tryplic diges-
tion on a larger scale, so as to study the products more fully. Two-
thirds fill a large flask (1 or 2 litres capacity) with 1 per cent,
sodium carbonate solution ; add the chopped white of a hard boiled
egg ; then some trypsin solution or pancreas extract ; and finally
some antiseptic this is essential since tryptic digestion is otherwise
accompanied by active putrefaction or bacterial decomposition, by
which evil-smelling products (indol, skatol, sulphuretted hydrogen,
etc.) are formed. After two or three days, filter t!.e liquid.

(a) The sediment or precipitate in the liquid contains tyrosin.
After filtering, dissolve a portion of the precipitate in dilute hydro-
chloric acid, end test with Millon's reagent the solution becomes
red.

(b) Acidify about 5 c.c. of the filtrate with acetic acid, then add
bromine water drop by drop a reddish colour appears, which
gradually deepens, then disappears as more bromine water is added.
When the colour is no longer deepened on adding bromine water

P. B. 4



50 CELL-CONTENTS AND CELL-WALLS.

add a few c.c. of amyl alcohol, and shake, then allow to stand
the amyl alcohol separates, coloured red or violet. This reaction is
due to the presence of the amino acid tryptophane.

(c) Concentrate some of the liquid to small bulk by heating on a
water bath ; after a day, examine the residue with the microscope
for crystals of leucin and tyrosin. The leucin is chiefly in
brownish spheres showing radiate and concentric markings, the
tyrosin in bundles or rosettes of long white needles.

(d) The leucin is also obtained as a sticky residue if the filtered
liquid is treated with alcohol until no more precipitate comes down ;
filter and concentrate the filtrate on a bath.

(e) Treat a portion of the filtered liquid with Millon's reagent,
which precipitates any proteins present ; filter, and boil the filtrate
^a red colour indicates tyrosin.

6O. Amino Acids and their Derivatives. The

amino compounds (amines, amino acids, amides), con-
taining carbon, hydrogen, oxygen, nitrogen, and in some
cases (cystin) also sulphur, may be formed either in con-
structive or in destructive metabolism. That is, they are
intermediate bodies formed either on the up-grade towards
protein, or on the down-grade from protein to simpler
bodies. In either case they are important for transloca-
tion, being soluble and diffusible. Many of these sub-
stances are present in plants e.g. asparagin, which is
abundant in seeds of Leguminosae. Asparagin (and other
amino compounds) combines with non-nitrogenous sub-
stances to form proteins ; it often accumulates in those
parts of plants where there is not sufficient non-nitrogenous
material at hand for the formation of proteins, Asparagin
may accumulate in plants which are grown in darkness, so
that photosynthesis cannot take place. Lupin seedlings
germinated in darkness contain a large amount of aspara-
gin, which disappears when the seedlings are placed in the
light. If, however, the seedlings are exposed to light in
an atmosphere deprived of carbon dioxide, the asparagin
persists in the seedlings. Both asparagin and tyrosin
occur in Dahlia tubers. Leucin is associated with aspara-
gin in seedlings of Lupin and other Leguminosae. In
Cruciferae, Cucurbitaceae, etc., asparagin is replaced by an
allied substance, glutamin.



CELL-CONTENTS AND CELL-WALLS. 51

(a) Make a strong aqueous solution of commercial asparagin, and
divide it into three portions. (1) Dissolve some copper sulphate in
water, and add dilute potash ; collect the precipitate on a filter,
and wash it with water. Add this precipitated copper hydroxide to
the asparagin solution asparagin (and other amides) gives a deep



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