Clinical Reviews in Allergy

@Copyright 1993 by Humana Press Inc,

0731-8235/93/325-337/$3.60

The Composition of NaturaJ Latex

from Hevea brasHiensis

Jean-Louis Jacob, 1 Jean d ' A u z a c l

and Jean-Claude Prev6t 2
1Programme Hevea (CIRAD-CD), B.P. 5035,
34032 Montpellier Cedex 1, France and 2Universite
de Montpetlier II, 34095 Montpellier Cedex 5, France

Introduction
Hevea brasiliensis is the source of "natural" latex. Latex
s y n t h e t i z e d in a network of laticiferous rings organized as
paracirculatory vessels systems, in the inner bark of the tree, is
collected by periodic incision of the bark (tapping). Latex is the
cytoplasm of the laticiferous cells; its organominerat composition
has all the features of ordinary plant cytoplasm except tha t it
contains 30-45% rubber and the nuclei and mitochondria are not
expelled during tapping. However, the major metabolism of these
cells is the conversion of sucrose into cis-polyisoprene, and the
major part of the enzymes involved in this process is located in
the cytosol, with a lot of other proteins. Unlike ordinary plant
cells, the cells that form the laticiferous vessels do not possess
large single vacuoles but a large numbe r of lysosomal micro-
vacuoles:tutoids. In addition to the organic and mineral solutes
with low molecular weight that are accumulated by active trans-
location, tutoids contain large quantities of anionic and cationic
proteins~ Some of these (heveamines) have chitinase/lysozyme
type enzymatic properties. Others, such as heveine, can bind to
chitin and are fungitoxic; hevein has considerable structural analogy

Clinical Reviews in Allergy 325 Volume 11, 1993

326 Jacob, d'Auzac, and Prevdt

with plant agglutinins (lectins) such as those in w h e a t germ and

Urtica dio~ca. The rubber particles themselves are s u r r o u n d e d
by phosphotipoproteins and glycoproteins; a m o n g t h e m an enzy-
matic protein involved in the elongation of the isoprene chains
w a s recently extracted, isolated, sequenced, and cloned. The s a m e
o p e r a t i o n s w e r e p e r f o r m e d on a g l y c o p r o t e i n t h a t is prob-
a b l y involved in latex coagulation t h r o u g h the agglutination of
r u b b e r particles.

The Laticiferous System

in Hevea and its Exploitation
Although n a t u r a l rubber, which is still indispensable in a
n u m b e r of major industrial sectors, can be synthesized by num-
erous plant species (1), it is currently produced only from the
processing ofHevea brasiliensis latex. Laticiferous tissue is found
in all p a r t s ofHevea brasiliensis trees, from the roots to the leaves.
It is organized in the phloem structures and much research has been
performed on its anatomical, histological, cytological, biochemical,
and physiological aspects, as reviewed recently by d'Auzac et al. (2).
It is exploited in the trunk, for reasons of convenience.
Rubber tree laticifers are articulated and a r r a n g e d as p a r a -
circulatory systems (3,4). The cambium r h y t h m i c a l l y creates a
monocellular layer t h a t t u r n s into a laticiferous ring. The cells of
which it is formed differentiate rapidly and become a n a s t o m o s e d
to form a syncytial continuum in which cis-polyisoprene very soon
becomes the main feature of the general metabolism, it should be
noted that there is no link between two successive laticiferous rings.
Figure 1 shows the location of the laticiferous tissue in the
i n n e r b a r k and the tissue "supply routes." A thin strip of func-
tional secondary phloem conveys photosynthates from the foliar
crown. The horizontal rows of cells are the m e d u l l a r y and liber
r a y s t h a t cross the cambium and convey w a t e r and m i n e r a l s from
t h e wood vessels to the cortical zones.
Micromanometric m e a s u r e m e n t s have shown t h a t t u r g o r
p r e s s u r e in the laticiferous rings reaches 0.9 to 1.5 M P a (5). This
is w h y t h e i r content (latex) is expelled -when the b a r k is cut w i t h
a gouge. The paracirculatory nature of the laticiferous rings accounts
for the continued flow and relatively large amount of latex collected

Clinical Reviews in Allergy Volume 11, 1993

Composition of Natural Latex 327

Fig~ 1. Hevea brasiliensis: a biological rubber factory (adapted from Hebant

and de Faij [1]).

(50-500 mL according to conditions) per tapping (the term used

in rubber-growing) per tree. After a varying length of time, latex
coagulates on the t a p p i n g cut a nd blocks the wound. The
laticiferous system then regenerates the lost celt material before
the next tapping in which the wound is reopened (generally a
slanting half-spiral). Tapping is carried out at regular intervals
(2 or 3 times a week).

Hevea brasiliensis Latex:

Cellular Cytoplasm
The Hevea brasiliensis latex collected during tapping is thus
the content of the cells articulated laticiferous system. Obser~a-
tions in situ and in vitro (4,6) using electron microscopy, high

Clinical Reviews in Allergy Volume 11, 1993

328 Jacob, d'Auzac, and Prevdt

speed centrifugation (7), and biochemical studies (2) have con-

firmed that it is a true cytoplasm.
Figure 2 represents diagrammatically the various constitu-
ent elements of a laticifer. However, it should be noted that nei-
t h e r n u c l e i nor m i t o c h o n d r i a are expelled d u r i n g t a p p i n g
b u t r e m a i n in situ because of their parietal position in more hya-
line, compact cell material. This phenomenon is of capital impor-
tance and enables the inter-tapping regeneration phenomena
mentioned above to function. The composition of all the major
subcellular compartments is summarized in Table 1 and exam-
ined in greater detail below.

The Nature and Composition

of Latex Organelies
Rubber Particles
R u b b e r p a r t i c l e s are the most n u m e r o u s organelles in
laticiferous cells. They form 30-45% of the total volume and 90%
of latex dry weight. They are generally spherical, but m a y be
ovoid or pear-shaped, and vary considerably in size between 60
and 5-6 ~m (6). They consist mainly of cis-polyisoprene, a poly-
mer with a high tool wt: 500-2000 kDa (2); the particles are enclosed
in a fine phospholipoprotein envelope whose electron negative
charge ensures the colloidal stability of the medium (8). The lip-
ids a n d p h o s p h o l i p i d s t h a t form this m e m b r a n e form 1.4-
3.2% of the rubber weight and contain 0.4-1.2% neutral tipids.
Their complex composition has been studied in fair depth (9).
Archer (10) characterized a globulin among the proteins
enveloping rubber particles. More recently, an enzyme involved
in the synthesis of polyisoprenic chains (the Rubber Elongation
Factor, REF) of 14.6 kDa (11,12) was sequenced and cloned
(12,13). Finally, a 23 kDa gtycoprotein that contributes to latex
coagulation by an agglutination mechanism has also recently
been sequenced and cloned (14).

Lutoids
Lutoids are small, 2-5-gin single-membrane vesicles (7).
T h e y form a polydispersed, l y s o s o m a l vacuole (15) a n d form
10-20% of latex volume (1). They are directly involved in cellular

Clinical Reviews in Allergy Volume 11, 1993

Composition of Natural Latex 329

LAT~CIFEROUS LEAVES
CELL
1

%
L,
SUCROSE
SUCROSE 4

ENERGY "~ ~ ~ '.'':.'Z']

+ |~.ol iONS ~..::!:..: ~ AM
-2 J~ t fMgl',<ily Ac

VASCULAR
pIEp.~-QORGAN,C AC.
RAY J
PYRUYATE 10NS
H20
IONS ~1 \ 9~ ~ AM AC
H20
CRUDE SIEVE

....... '1' ....

~LYSOMES SIEVE
TUBE

RNA

FLOW Z~ LEUS
444,,

ENOLS

Fig. 2. Scheme of Hevea brasiliensis laticiferous system. Shown are its sucrose,
water, organic, and inorganic elements loading via medullary rays and sieve tubes,
its different cellular organits, and the major biochemical pathway leading to the
polyisoprene (rubber) synthesis (adapted from Jacob) (1).

Clinical Reviews in Allergy Volume 11, 1993

 Table 1
Organic N o n r u b b e r Constituents of Latex from H e v e a b r a s i l i e n s i s ~
-- Protein
0.5 ~
-- P h o s p h o l i p i d s
0.6
_ Rubber -- Tocotrienols (free
phase a n d esterified)
37 0.09
- - Sterols and sterol
esters
- - Fats and waxes

Frey- ~ Carotenoids
- Wyssling Plastochromanol
particles O t her lipids

- Inositols
-

1.0-1.5
-- Carbohydrates
-- Proteins
0.5
--Glutathione
0.01
F r e s h latex _ Serum - a m i n o acids
- F r e e

48 0.08
-- Ascorbic acid
0,02
-- O t her organic acids
- - Nitrogenous bases
0.04
- - Nucleic acids
0.002
- - Mononucleotides
0.02
- - Proteins
0.2
-- P h o s p h o l i p i d s
Bottom - -Plastoquinone
- fraction -- U b i q u i n o l
15 - -Sterols
- -Trigonelline
0.007
- -Ergothioneine
0-0.05
"Adapted from Archer et al. (27).
ZThe figures under the components indicate their approximate concentra-
tions in g/t00 g of tatex.

Clinical Reviews in Aflergy 330 Volume 11, 1993

 Composition of Natural Latex 331

homeostasis of the laticiferous system (16) and play a major role

in the process leading to latex coagulation (8).
Their membrane, which can be considered as tonoplast, is
remarkably rich in phosphatidic acid (17) and therefore has a
strong negative charge. Several enzymes have been character-
ized in this m e m b r a n e , such as an A T P a s e functioning as
an influx proton p u m p (78,19), a pyrophosphatase (20), and a
N A D H - c y t o c h r o m e c-reductase that can function as an outgoing
p u m p for protons accumulated in the vacuole (76,19).The finding of
antiports between protons on the one hand and citrate,basic amino
acids, and calcium on the other (21) implies the existence of the cor-
responding proteinic transporters.
Vacuole contents are acidic (pH 5.5) (15). Seven to eight ani-
onic and cationic intralutoid proteins were found from 1960
onward (22,23). S o m e are arranged as structurally complex
microhelices whereas others are microfibrils (7). Hevein, an ani-
onic protein, is quantitatively the most important (70% of total
proteins). The m o n o m e r protein containing 43 amino acids (5
kDa) (24) has been sequenced and cloned (14,24,25); it is particu-
larly rich in cystein and glycine and also binds to chitin, showing
t h a t it has a strong antifungal activity in vitro (26). It would
appear today that herein is involved in one of the mechanisms
that causes latex to coagulate by agglutination with the 23 kDa
protein in rubber particles (14). Herein has strong structural
homology with the lectins specific to N-acetylglucosamine, such
as the agglutinins in wheat, barley, and rice germs, and with
several proteins induced by wounding in potato (25). Homology
has also been demonstrated with Urtica diofca agglutinin (26).
Two major basic proteins--heveamines A and B--have been
isolated from lutoids (27). They display strong homology through
their amino acid contents with the lysozymes in ficus and papaw
latex (28) and their suspected chitinase activity has since been
confirmed (29).
In addition to these quantitatively important proteins, the
lutoids (lysosomal vacuoles) contain numerous acid hydrolases
characteristic of animal lysosomes (acid phosphatase, manno-
sidase, RNase, DNase, and so on) as well as peroxidases (15,30).
Analysis of solute distribution between eytosol and vacuole (Tabte
2) shows that these organelles strongly accumulate certain celt
Clinical Reviews in Allergy Volume 11, 1993
332 Jacob, d'Auzac, and PrevSt

Table 2
Cytosolic and Lutoidic Sera Compositions
of Latex from Hevea brasiliensis
Concentrations (mM)
Ratio
Lutoids lutoids/
Solutes (vacuoles) Cytosol cytosol
K§ 31.2 30.1 1.0
Mg 2+ 64.2 8.3 8.0
Ca 2+ 1.5i 0.25 6.0
Cu 2~ 0.047 0.021 2.0
Pi 76 9.1 8.7
Sucrose 5.8 40.5 0.]_
Citrate 53.0 5.7 9.3
Malate 17.3 14.6 1.2
Amino acids
Acidic 22.9 56.9 0.4
Neutral 21.1 36.4 0.6
Basic 56.9 6.6 8.6
Adapted from d'Auzac and Jacob (32).

elements, such as Mg 2+, citrate, Pi, Ca 2+, and basic amino acids.
However, they contain very little sucrose, and their K + and malate
contents are similar to that of the cytosol. This characteristic,
resulting from the functioning of the lutoids, highlights their role
in cell homeostasis (21).

Frey Wyssling Particles

These 5-6-~m organelles, discovered by Frey Wyssling (31),
are much less numerous than the lutoids (2-3% of latex volume)
(2). They are limited by a double membrane and have an
extremely complex structure similar to that of certain chromoplasts
(7). They contain lipid globules, isoprenic compounds, plasto-
chromanols and plastoquinones, and carotenoids responsible for
their orange color.
The fact that they contain o-diphenoloxidases strengthens
the analogy with plasts (32). Some authors have considered that
these o-diphenoloxidases may be involved in the coagulation with
latex in contact with air during tapping (2) that destroys a lot of
Frey Wyssling particles. Nevertheless, the biological role of these

Clinical Reviews in Allergy Volume 11, t993

Composition of Natural Latex 333
organelles in situ has not been clearly defined, although some authors
consider that they may be involved in isoprenic synthesis (2).
Nucleus and Mitochondria
We remind you that no trace of nuclei or mitochondria is
found in latex collected by tappings. However, ribosomes and
polyribosomes have been found in latex (7), and their functions
in protein synthesis have been studied (33). Messenger RNA has
been extracted from latex and translated in vitro (34).
Cytosol
Organelles can easily be separated from the cytosol by ultra-
centrifugation (7). The cytosol forms 40-50% of latex volume and
is the site of the greater proportion of rubber synthesis and the
general metabolism (2).
Cytosol Minerals
K + has the highest level (Table 2) and its role as in situ
osmoticum should therefore be stressed. It is followed by Mg 2+,
inorganic phosphorus (Pi), and then much smaller amounts of
Ca 2+ and Cu 2§ (Table 2). Expressed in mg/100 g of latex, the Pi,
Mn, Fe, and Zn levels average 7, 0.1, 2, and 0.3 respectively.
Soluble Carbohydrates
Sucrose is the initial molecule in isoprenic synthesis and the
m a i n element in the laticiferous metabolism (35,36). The level
m a y v a r y considerably (from 1 to 40 raM) according to the condi-
tions. It is the most plentiful glucid in latex; there is also a little
glucose (1 raM), fructose ( <1 mM) and raffinose (<1 raM) (32).
The presence of relatively large a m o u n t s of quebrachitol
(monomethyl-l-inositol) should be underlined (t% of the weight
of fresh latex). Like K § and sucrose in certain cases, this polyol
m u s t play a nonnegligible role as osmoticum in the laticifers (37).
Organic Acids
These are numerous in the cytosol. However, only citrate and
malate are found in fairly high concentrations: 5-20 mM (Table
2). The others (acetic, formic, propionic, lactic, pyruvic, succinic,
furnaric, and acon2tic acids, and others) are found as traces, reflect-
ing the functioning of an active intermediate metabolism (38).

Clinical Reviews in Allergy Volume 11, 1993

334 Jacob, d'Auzac, and PrevSt

Amino Acids
Distribution between basic, neutral, and acid groups is shown
in Table 2. Glutamate, glutamine, alanine, and aspartic acid are
quantitatively the most important and form 81% of total cytosol
amino acids (39).
Nitrogenous Bases
Some authors (27) have reported the presence of nitrogen
bases, such as methylamine, tetramethylene-diamine, penta-
methyl-diamine, trigonelline, and stachydrine. Etha nola mine is
mentioned by Brzozowska-Hanower et al. (40).
Reducing Agents
Reducing agents play a major antisenescent role in the cells
(16). Thiols are mainly represented by glutathion, found at a con-
centration of about 1 mM and cysteine, whose level does not
exceed 0.5 mM (40). The ascorbic acid content is 2-5 mM (32).
Nucleotides and Nucleic Acids
Phosphate nucleotides are represented m a i n l y by phosphate
adenosines (<0.5 raM), but phosphate uridines (including UDPG)
and phosphate cytidines are also found (32). Although the NAD
content has been assessed at about 40 t~//, no NADH or NADP(H)
has been detected (41). RNA and the relative proportions oftRNA
and rRNA have also been studied (42). However, no trace of DNA
has been detected, which confirms the retention of nuclei and
mitochondria in situ during tapping.
Calmodulin
It should be noted that this molecule, classically involved
with Ca 2+in intracellular regulation, has been found in nonnegli-
gible quantities in latex cytosol (43,44).
Proteins
Cytosol proteins are extremely numerous, forming 0.6% by
weight of fresh latex. Some may play an allergenic role (23). A
relatively abundant globulin whose isoelectric point is 4.55 was
found by Archer et al. (27). It appears to be able to combine closely
with the envelope of rubber particles and thus affect the colloidal
stability of latex. Nearly 100 enzymes involved in the general

Clinical Reviews in Allergy Volume 11, 1993

 Composition of Natural Latex 335

metabolism and isoprenic anabolism have been characterized and

studied (30) in cytosol. The very numerous cytosol enzymes found
(30) reflect the intense metabolic activity of laticifer cytosol.
Attention is d r a w n in particular to the presence of three differ-
ent proteins that have been named hevaine a, b, and l in the
cytosol and the lutoid serum and that behave as proteases (45).
Hevaines are present in commercial natural latexes.

Conclusion
Hevea brasiliensis latex collected by t a p p i n g can be consid-
ered as cytoplasm, in spite of the absence of nucleus and mito-
chondrial material, tt possesses the complexity of cytoplasm both
in its subcellular organization and the diversity of the constituent
molecules. Its essential characteristic is the high rubber content.
Rubber is a very long chain cis-polyisoprene polymer synthesized
in the cytosot compartment. Rubber forms 90% of latex dry weight
and 40% of fresh weight, which underlines the astonishing domi-
nance of this anabolic p a t h w a y in the laticiferous metabolism.
However, the p r e p a r a t i o n of dry rubber or concentrated latex as
used in i n d u s t r y removes a fairly large proportion of the mineral
and organic solutes from fresh latex. Latex processing conditions
in p l a n t a t i o n factories can considerably modify the technological
properties of r u b b e r after vulcanization t h r o u g h the influence of
the n a t u r e and proportion of the residual n o n r u b b e r substances.
Some of these give n a t u r a l rubber properties t h a t are still better
t h a n those of synthetic cis-polyisoprene.
In contrast, it appears to have been clearly d e m o n s t r a t e d
t h a t the proteins extracted from latex or from rubber tree leaves
containing latex display allergenic properties (46,47). T u r j a n m a a
et al. (48) observed t h a t allergenic proteins extracted from latex
gloves have molecular weights of between 2 and 30 k D a and are
t h u s similar to various allergens, including those of pollen. It h a s
been mentioned t h a t large quantities of proteins with low molec-
u l a r weights are found in latex.

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Clinical Reviews in Allergy Volume 11, 1993

336 Jacob, d'Auzac, and Prev6t

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 Composition of Natural Latex 337

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Clinical Reviews in Allergy Volume 11, 1993