The chemical fractionation of rabbit and swine thymus.
ABSTRACT A chemical fractionation procedure, previously found applicable to bovine thymus and bovine and ovine palatine tonsils, was used to fractionate rabbit and hog thymus. With respect to the chemical fractionation steps, yields of fractions, and optical and electrophoretic properties, extracts from hog and rabbit thymus were indistinguishable from similar extracts prepared from calf thymus. The study provides composition and yield data applicable to the thymus of a small mammal readily available in most laboratories.
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ABSTRACT: The extraction behavior of thymus and the composition of fractions prepared from this organ has been studied. Sequential extraction methods using 0.15 M NaCl followed by water gave information with respect to the weight fraction of cytoplasmic and nuclear constituents. Lipide, nucleic acid, and electrophoretic analysis of the extracts provided additional information. A less complex electrophoretic pattern was obtained from subsequent extracts in the sequence. Sucrose and saline dispersates obtained from tissue fragmented with either the Potter-Elvehjem homogenizer or in a Waring blendor were fractionated, using standard differential sedimentation methods. The fractions obtained by means of four different dispersion procedures were compared in terms of yield, chemical analysis, and electrophoretic composition. The quantity of material in thymus having the sedimentation characteristics of liver mitochondrial and microsomal fractions was remarkably small. Both the suspension medium employed and the method used to bring about a disruption of the cells in the tissue affected the yield of "particulate" material. The components present in the later extracts in the sequence, E(4) to E(7), in the case of sequential extraction study resembled with respect to chemical composition and electrophoretic characteristics, the microsome fraction prepared by differential sedimentation methods. About 76 per cent of the PNA in the tissue appeared to be in the cytoplasm. The remaining 24 per cent PNA was found in the nucleus and accounted for 1.7 per cent of nucleus on a dry weight basis. From 75 to 88 per cent of cytoplasmic PNA was extracted from the tissue and 76 to 94 per cent of the PNA in the extract was found in the final supernatant solutions, depending upon the dispersion methods and suspension medium used in the extraction procedure. The composition of the final supernatant fractions using differential sedimentation methods were comparable in terms of electrophoretic properties, protein concentration, nucleic acid content, and fractionation behavior to saline extracts E(1) to E(3), of thymus used in earlier studies.The Journal of biophysical and biochemical cytology 12/1958; 4(6):717-25.
812 BRIEF NOTES
The Chemical $'ractionation of Rabbit and Swine Thymus.* BY EUGENE L.
HESS AND SAIMA E. LAGG. (From The Rheumatic Fever Research Institute,
A previous report described a gen-
eralized method for the chemical frac-
tionation of lymphatic organs (1). It was
shown that the method was applicable to
bovine thymus and to ovine and bovine
palatine tonsils. The study has now been
extended to include two additional spe-
cies, in order to ascertain the general
applicability of th~ method, and also to
include an animal common to laborato-
ries. The procedure would be much more
useful if applicable to the thymus of a
small mammal such as the rabbit, since
relevant biological studies become more
feasible, and numerous biological prob-
lems could be undertaken using data from
normal animals as a frame of reference.
Problems such as the radiation sensitivity
of lymphoid tissue (2), the antibody con-
tent of lymphatic organs (3), and the
changes in composition resulting from
the involution of the thymus under con-
ditions of stress (4), appear to be sus-
ceptible to study using chemical fraction-
ation procedures. It is the purpose of this
report to point out that the fractionation
procedure previously described (1) has
been found equally applicable to porcine
and rabbit thymus.
Materials and Methods
The thymus glands were procured from
young animals, packed in ice, and the extra-
neous tissue removed within 2 hours from
the time of slaughter. All experimental opera-
tions have beeen discussed previously (1, 5).
The preparation of extracts and symbols used
to represent the various fractions are the
* Supported by a grant from the Atomic
Energy Commission, Contract AT(11-1)366
with The Rheumatic Fever Research Insti-
tute, and by the Leukemia Society.
Received for publication, May 13, 1957.
same as used previously (1). The term
optical concentration was defined in the
previous study (1).
In our experience rabbit thymus con-
tained a much larger amount of fat dis-
persed throughout the organ than did
porcine and bovine thymus. This was
evident even with young rabbits carefully
selected with respect to age and size. Al-
together from 100 gm. of starting material,
representing the thymus glands from 18
rabbits, 42 gm. of fat were removed by
hand from cold thymus and an additional
11 gm. floated to the surface of the extracts
after centrifuging. As a consequence only
47 gm. of non-fat-containing material was
used in the rabbit experiment. The results
have been expressed, however, in terms of
100 gm. wet tissue containing no fat.
As can be seen in Fig. 1 A the electro-
phoretic pattern obtained from the total
extract (ET) using rabbit thymus was
indistinguishable from the previously
thymus. The pattern from hog thymus
is virtually identical with that seen in
Fig. 1 A.
In each subsequent fraction examined
(5.1 P, 5.1 S, 3.0 P, 3.0 S, 6.2 P, 6.2 S,
4.7 P and 4.7 S) the electrophoretic pat-
terns closely resembled those published
in the earlier study (1). For comparison
purposes patterns obtained using the
PNA-type nucleoprotein fraction 3.0 P
and fraction 4.7 S from hog thymus are
shown in Fig. 1 B and 1 C respectively.
In all cases yields of each fraction, on
a dry weight basis, were comparable to
those reported in the earlier study (1).
The optical concentrations and yields
are listed in Table I. The materials re-
maining insoluble after three extractions
(1) from bovine
J. BioPltYsle. AND BlOC~E~. CYI'OL., 1957, Vo]. 3, No. 5
BgIEF ~OTES 813
Fro. 1. Electrophoretic patterns (descending limb) from thymus extracts, after 120 minutes
under a potential gradient of 6.4 volt cm. -1 in veronal buffer/z 0.10, pH 8.6. Magnification
factor from cell to print is 1.02. Ascending patterns are essentially enantiographs of the patterns
shown. A. Pooled extracts of rabbit thymus, protein concentration approximately 0.85 per
cent, diagonal slit angle 50 °. B. Fraction 3.0 P (PNA-nucleoprotein) from swine thymus.
Protein concentration approximately 0.6 per cent, diagonal slit angle 60 °. C. Fraction 4.7 S
from swine thymus. Protein concentration approximately 1.2 per cent, diagonal sift angle 40 °.
amounted to 9 gm. as in the case of the
earlier study (1).
tion procedure can, therefore, be recom-
mended as a possible tool for biological
Optical Concentrations (0. C.) and Yields of Hog and Rabbit Thymus Extracts
and Fractions per 100 gin. Wet Tissue
* Optical concentration represents optical density times dilution at the wave length
:~ Chiefly nuclei and connective tissue remaining insoluble after three extractions with
0.15 M NaC1.
The chief point of interest in the above
study was to compare the macromolecu-
lar composition of thymus from several
animal species. It is of interest, both from
an academic and a pragmatic viewpoint,
that according to the chemical and
physical methods employed in this study
and in the earlier report (1) hog, calf,
and rabbit thymus extracts were indis-
tinguishable. One can infer that the
chemical fractionation procedure em-
ployed would be applicable to thymus
from other common mammals. It also
seems reasonable to suggest, as has been
shown for palatine tonsils and thymus,
that the method may apply to other
lymphatic tissue as well. The fractiona-
problems where macromolecular com-
position is relevant to evaluation of
results. The composition and yield data
given above for rabbit thymus provide
a convenient frame of reference applicable
to a small mammal readily available in
The absence of color when rabbit and
hog thymus extracts were heated with
diphenylamine reagent provides further
evidence that the mucoprotein found in
palatine tonsil extracts was not a lympho-
cytic constituent (1, 5).
A chemical fractionation procedure,
previously found applicable to bovine
thymus and bovine and ovine palatine
tonsils, was used to fractionate rabbit and
hog thymus. With respect to the chemical
fractionation steps, yields of fractions,
and optical and electrophoreticproperties,
extracts from hog and rabbit thymus
were indistinguishable from similar ex-
tracts prepared from calf thymus. The
study provides composition and yield
data applicable to the thymus of a small
mammal readily available in most
1. Hess, E. L., and Lagg, S. E., J. Biophysic.
and Biochem. Cytol., 1956 2, 557.
2. Cronkite, E. P., and Brecher, G., Ann.
New York Acad. Sc., 1955, 59, 815.
3. Coons, A. H., Ledue, E. H., and
Connolly, J. M., Ann. New York Acad.
Sc., 1955, 59, 951.
4. Gordon, A. S., Ann. New York Acad. Sc.,
1955, 59, 907.
5. Hess, E. L., Ayala, W., and Herranen,
A., J. Am. Chem. Soc., 1952, 74, 5410.
J. BIOI~Yslc. AND BIOCI~K. CXTO~., 1957, Vol. 3, No. 5