- Email: [email protected]
Transfer factor: Hypoxanthine is a major component of a fraction with in vivo activity
Transfer factor: Hypoxanthine is a rn component of a fraction with in viv activity Russell H. Tomar, Syracuse, N. Y.
M.D.,
Ruth Knight,
MS.,
and Malvin
Stern,
Transfer factor was prepared from the leu,kocyte lysates of four donors with known skin test reactivity. After ?Lltrafiltration and do&&-gel filtration on polynoylumide gels, fraction IV of the preparation was found to have ‘biologic activity. This fruction contained one major and ocoasionully one minor &raviolet-absorbing a.& zero to on.e ninkydrin-detectable sports on thin-layer chromatography. The major ultraviolet spot was identified as kypoxanthine. Hypoxnnthine was demonstrated to be responsible for the high 260 nm/280 nm ratio of preparations with 6iologi.o activity in viuo. It wus not determined if hypoxanthine is reqzcired for transfer factor activity. 1n addition, an orctiol-negative preparation also had biologic activity.
Human leukocyte lysates contain low molecular weight material which has the ability to transfer to a recipient the delayed skin reactivity of the donor. This material has been called transfer factor (TF) by Lawrence1 and his eel,leagues. The existence, specificity, and small size of transfer factor has been confirmed many times. There is, however, little knowledge about its biochemical composition.ls 2 Baram and associates semipurified a material which appeared to be composed primarily of nucleic acid bases. Kirkpatrick and Galliq2z 5 Neidhart and eolleagueq4 and GottliebF have described preparations which elute as low molecular weight materials on Sephadex columns. In this communication, we describe the use of ultrafiltration and polyacrylamide (P-2) gel filtration to prepare transfer factor and thin-layer chromatography and spectrophotometrie scanning to analyze it. TERIALS AND In vivo testing
METHODS
Because of ethical consideration, normal human volunteers were not used. Therefore, transfer factor preparations were tested on two subjects with immunodeficiencies. Other modalFrom the Department of Pathology? Division of Clinical Pathology, Department of Medicine, Upstate Medical Center, State Umversity of New York. Supported in part by General Research Support Grant RR05402 from the Division of Res#earch R.esources, National Institutes of Health. Presented in part at the meeting of the American Association of Immunologists, Atlantic City, N. J., April, 1975. Received for publication Aug. 15, 1975. Accepted for publication Dec. 1, 1975. Reprint requests to: Dr. Russell H. Tomar, M.D., Upstate Medical Center, State University Hospital, 750 East Adams St., Syracuse, N. Y. 13210. Vol. 58, Wo. 1, Part 8, pp. 190-197
Transfer
VOLUME 58 NUMBER 1, PART 2
TAELE I. Preparation
ik:. EL
of human
iti M M
transfer
2: t:
factor:
42 x42 15x20 30x40 50x50
factor
%
Skin test of donors
22x35 11x18 17x20 10x11
7i “t;’ ci
ii is Y0
‘One-tenth milliliter injected intradermally. t SK-SD 100/25 U/ml (Lederle) . $Can&da 1: 50 (IIollister-Stier ), $PPD 5 TU (Connaught). /jHistoplasmin (Parke, Davis & Co.). ities of therapy had failed in both of these patients, The first had chronic mucocutaneous candidiasis and the second had ataxia telangiectasia. Both had negative skin reactions to 0.1 mi intradermal injections of etreptokinase-streptodornase (100/25 units per ml, Lederle) and Candida (l-50, Hollister-Stier) and a decreased number of circulating lymphocytes dlieh spontaneously formed rosettes with sheep erythrocytes (T cells). Transfer factor was tested in two ways as described by Lawrence.1 In the first-the local assay-materials were adjusted to an absorbance at 260 nm of 0.200 to 0.300, i.e., 5% to 10% of the absorbance in Fraction IV. Aliquots of 0.1 ml were injected intradermxlly along the volar aspects of the forearms along with a buffer control. Tlventy-four hours later, antigens to Fvhich the donor of the transfer factor was sensitive mere injected onto the same areas as previously injected with the transfer factor preparations. Reactions were read at 24 and 48 hr after the introduction of antigen 1)~ two observers one of whom had no knowledge of this protocol. All fractions could be tested at the same time in this way. The second procedure for testing transfer factor-the systemic assay-required adjusting absorbance at 260 nm to 0.800 to 1.00. An nliquot of 0.2 ml was injected intradermally. Twenty-four to 48 hr later, antigens were tested in the contralateral ext,remity. Only a single fraction could be assayed at any one t,ime. Again, skin tests were read at 24 and 48 III. by two observers one of whom was aware of the protocol. In addition, spontaneous erythrocyte rosetting by peripheral lymphcytes was determined before rind after transfer factor injection.7, 8 Preparation
of transfer
factor
h unit of blood was drawi from donors selected for their skin reactivity to SK-SD, Candidn, PPD (Connaught), and histoplasmin (Parke, Davis) (Table I). After the buffy coat containing l-2 x 109 leukocytes TX-as removed in 20 to 50 ml of plasma, it was frozen and thawed 4 to 10 times before ultrafiltration with positive pressure (Amicon X-MSO). The filtrate was then gel filtered through a 50 x 1.5 cm polyacrylamide P-2 (Biogel) column with ammonium carbonate buffer (pH 7.4, 0.001 M) (Fig. 1, a). Flow rate was 30 to 40 ml per hour and 3 to 5 ml was collected in each tube. Absorbances at 260 and 280 nm mere determined. The fractions in the individual peaks were pooled and refiltered through the same column (Fig. 1, B). The refiltered peaks were used for further testing and analysis. Approximately 57’ of the original absorbance units at 260 nm were present in the refiltered Fraction 1V. iochemical
analysis
Phosphates mere determined by converting condensed phosphates to orthophosphates with sulfuric acid and determining the phosphate concentration by the reduction of phosphomolyhdic acid with ascorbic acid. ATP adjusted to an optical density of 0.733 and 1.382 at 260 nm (0.045 and 0.090 mg/ml, respectively) was used for reference.9 Ribose was estimated by the orcinol reaction using adenosine, inosine monophosphate, 2’3’ guanosine monophosphate, and cyclic cytidine as references.10 Absorbance maxima mere determined with a recording scanning spectrophotometer (Beck-
192
J. ALLERGY CLIN.
Tomar, Knight, and Stern
---
IMMUNOL. JULY 1976
260nm 280nm A RECHROMATOGRAPHY M.K.IP:
.4 ------
260 mm 260 mm
.3
Tube #
IO
20
30
40
50
Q FIG. 1. A, The ultrafiltered leukocyte lysate was gel filtered through a polyacrylamide (P-21 column of 50 x 1.5 cm using ammonium carbonate (pH 7.3, 0.001 M) as buffer. Flow rate was 30 to 40 ml/hr; samples of 3 to 5 ml per tube were collected and absorbances at 260 nm and 280 nm determined. The tubes comprising the major peaks were combined and dried from the frozen state. Blue dextran eluted at tube 10. 8, The combined (yophilized material was brought up in the same buffer and refiltered on the same column. Shown is a typical refiltration of Fraction IV. The tubes noted by the black bar were combined and used for further testing. man Model 25). Primary amino acids were detected with fluorescamine using phosphate buffer (pH 8.3, 0.067 M) and 10 mg of fluorescamine in 100 ml dioxane.11 Thin-layer chromatography was done on silica G (F2.54) plates (250 p) with butanol:acetic acid:water 4:l: 1 as solvent and on cellulose plates (100 p) with methanol:hydrochloric acid:water 7:2 :1 as solvent. A closed atmosphere system was used. The ascending front was allowed to develop 100 mm. Forty to 60 ~1 of concentrated material (A260 nm = 0.80 to 3.5) were applied. Ultraviolet absorption (254 nm) for nucleic acid constituents, ninhydrin spray for primary amino acids, peptides and proteins, fluorescamine spray for the same materials, and carbon charring with sulfuric acid were used as methods of detection.12 Standard bases, nucleosides, and nucleotides for scanning and thin-layer chromatography were obtained commercially (Sigma) and adjusted to approximately 100 to 200 pg/ml. Xantbine oxidase (Sigma) was adjusted to 0.12 U/ml in phosphate buffer (pH 7.5, 0.067 M). One-tenth milliliter of this enzyme was added to 0.9 ml of Fraction IV and incubated at 37’ 6.13 Spectrophotometric scanning was done before and during incubation. When the reaction was complete, thin-layer chromatography was performed on the treated material as well as on untreated Fraction IV.
Transfer factor activity was demonstrated to be present in Fraction IV. Delayed reactions, although small, were seen only where Fraction IV had been
VOLUME 58 NUMBER 1. PART 2 TABLE
II. “Local”
Transfer
assay
for transfer Patient
1
Patient
3x4 0
5x5 0
4x5 0
2x2 0
0
0
2x2 6x8 0
0 4x5 0
“One-tenth milliliter of material adjusted to 0.200-0.300 absorbance with antigen at the same site 24 hr later. tIndicates donor of transfer factor.
TABLE III. “Systemic”
assay
2
(TF,wt) (mm induration)
0 0
IV Buffer
1
factor
(mm induration)
II ITT
factor
for transfer
8X8
0
3x3 0
at 260 nm before
challenge
factor Peripheral lymphocyte sheep erythrocyte rosettes
January 21 February 4 March 4 March 18 March 29 April 18 April 22 May 8 June 20 June27 August 12 “Indicates iIndicates
0 II (MF*)
0
i O/O?
III (MF*) o/o
IV (MF*)
o/o
2:
6x8 8x10
8x8 10x10
o/o o/o
67 50
10x10
10x12
8x10 8x10 Normal
32260
IV (ww*)
donor of TF. two observers.
previously injected in the LLlocal” assay in both patients (Table II). In the systemic assay, Fractions II and III failed to convert the recipient from negative to positive ; while two preparations of Fraction IV allowed the previously negative recipient to develop skin tests of at least 6 mm induration. Incidently, the number of peripheral lymphocytes which bind sheep erythrocytes appeared to have increased after one patient had been treated with Fraction IV and perhaps III (Table III). Furthermore, this patient’s ungual moniliasis cleared. Results were similar on both thin-layer chromatography systems used. Therefore, only the silica G system results will be described.’ All four of the crude TF preparations contained 3 to 4 ninhydrin and 2 to 4 ultraviolet-detectable spots. Fraction II from all four subjects demonstrated 2 ultraviolet-absorbing spot,s of approximately the same Rf and a large homogeneous ninhydrin-staining area. All Fraction III preparations showed a ninhydrin-staining spot of similar Rf. All demonstrated a second substance-either ultraviolet absorbing (two samples) or ninhydrin staining (the other two samples). All four of the Fraction IV preparations showed one clearly identifiable ultraviolet-absorbing spot. Three of the preparations also contained a second faint ultraviolet-detectable area and a small pale ninhydrin-staining spot (Fig. 2).
194
Tomar,
Knight,
and
J. ALLERGY CLIN.
Stern
IMMUNOL. JULY 1976
//// m = absorbance at 254nm @$ @&$$= ninhydrin detectable
I III
I II
/ EL
FIG. 2. Thin-layer chromatography was done on silica G (F254) coated plates of 250~ thickness. Butanol:acetic acid:water (4:l :l) was the solvent. Twenty to 60 PI of material with an absorbance at 260 nm of 0.800 to 3.5 was applied. The front was allowed to move 100 mm in a closed atmosphere system. Ultraviolet absorbance and ninhydrin were used as methods of detection. Fractions II, III, and IV from a typical run are shown. Fraction III shows only ninhydrin-detectable material; Fraction IV primarily ultravioletabsorbing materials. TABLE IV. Thin-layer
chromatography Substance
Hypoxanthine Guanosine 2’3’ GMP IMP Uracil TF IV MF TF IV MK
[Silica
G [F 2541;
butanol:acetic
acid:li,0
[4:t:I
j]
Rf
43 33 ii :: 44
Spectrophotometric scans of crude material, &-actions II, III, and IV showed peaks of 252, 250, 290, and 249 to 252, respectively (Fig. 3). Scans of the four Fraction IV preparations were quite similar, peaking at 250 to 252 with a 260,’ 280 ratio of 3.0 to 5.1. A number of different bases, nucleosides, and nucleotides were surveyed by spectrophotometric scanning and by thin-layer chromatography and compared to Fraction IV. By scanning, only guanosine, 2’3’ guanosine monophosphate, hypoxanthine, inosine monophosphate (IMP), and uracil demonstrated maximal absorbance near that of Fraction IV. None, however, was superimposable upon Fraction IV. By thin-layer chromatography, hypoxanthine, but not IMP, migrated in a location similar to Fraction IV (Table IV). Xanthine at similar concentrations was not detectable by ultraviolet lamp. The major ultraviolet spot in Fraction IV was no longer demonstrable after treatment with xanthine oxidase-an enzyme which converts hypoxanthine to xanthine and xanthine to uric acid. This proeedure was repeated in three preparations. Xanthine oxidase treatment also shifted the absorbance maxima of the Fraction IV preparations tested, changing the 260/280 ratios from 3-5/l to l/1-3 (Table V).
Transfer
VOLUME 58 NUMBER 1, PART 2
factor
1
252
nm
230
300
230
360 251
290
FRACTIONIU -I!!!!&
FIG. 3. Scanning was done with a double-beam spectrophotometer Scans of the precolumn pool with transfer factor and Fractions typical preparation are shown.
TABLE V. Transfer
factor
I Wavelength (mm)
TF IV (MF)
293 250 2601280
with I
+x0 (55 min)
Alone
230
“Relative
IV treated
xanthine
oxidase
TF IV (MP) Alone
(Beckman II, III, and
Model 25). IV, from a
(X0)* I
+x0 (75 mm)
Hypoxanthine
z!x
XanthinexUric
acid
::*
ii
100
100
ii.2
z:
200
20 2.00
26 0.73
7 2.00
1;: 0.33
0 12.40
22 1.oo
1:: 0.36
absorbance
units.
Inorganic phosphates were determined on samples adjusted to an absorbance of 0.800 at 260 nm. Fraction II had greater than six parts per million phosphate; Fraction III had no detectable phosphate; and Fraction IV had 0.6 parts per million phosphates. ATP with an absorbance of 260 nm of 0.733 contained 2.4 parts per million phosphates. Buffer contained none, Thus, Fraction IV had approximately one fourth the amount of phosphate as concentrations of ATP giving similar absorbance at 260 nm. The amount of orcinol-positive material in Fraction IV varied greatly, indicating that the amount of ribose in each preparation also varied (Table VI).
19
Tomar,
Knight,
TABLE VI. Transfer
1:: IV
(Az,anm= 0.850)
J. ALLERGY CLIN.
and Stern
factor:
3 +
IMMUNOL. JULY 1976
Summary
2.50 290 250
Numbers in parentheses are number of preparations *ATP of approximately same A, = 2.4 ppm. tOrein assay. $Fluorescamine assay compared to phenylalanine.
6.0 None 0.6 tested.
Fraction IV was analyzed for primary amino acids by using fluorescamine. Compared to a phenylalanine standard, there appeared to be 2 to 3 @g/ml of primary amine-containing materials in preparations with an absorbance at 260 nm of 0.850. The amount of primary amino acid in the Fraction IV samples tested was relatively consistent (Table VI). DISCUSSION Fraction IV of our transfer fraction preparation is similar in many ways to previously described preparations. l-6 It elutes as a small molecular weight material by gel filtration and transfers in a delayed fashion to an intradermal antigenic challenge. Neidhart and associates and Kirkpatrick’s preparations eluted late from Sephadex gel columns and had a high 260/280 ratio and also transferred skin tests. Baram and colleagues’ small molecular weight transfer factor peaked at 258 nm ; Fraction IV peaks at 249 to 252. Baram’s group did not describe paper chromatography before acid hydrolysis; Fraction IV has one major ultraviolet-absorbing spot prior to hydrolysis. After hydrolysis, Baram and assoGates3 found three ultraviolet-absorbing spots which corresponded to adeniae, guanine, and cytidylic acid. They found no ninhydrin-detectable materials and neither did we in several of our preparations. The fluorescamine determination for primary amino acids, peptides and proteins has not been performed on previously described preparations. It is more sensitive than ninhydrin. I1 It is interesting to note that the primary amino acid content of different TF IV preparations was relatively constant while orcinolpositive material varied greatly, Since we do not know the relative activities of our materials, we cannot calculate specific activities on the basis of either amino acid or ribose determinations. The high 260/280 ratio and positive orcinol tests have been the chief reasons for believing that transfer factor contains oligonucleotides. However, it appears as if hypoxanthine and perhaps xanthine-probably as preparative contaminants -give TF its absorbance characteristics. Moreover, our preparations had a variable amount of orcinol-positive material including some with in vivo activity with no detectable ribose. Since we were unable to test our materials after treatment with xanthine oxidase, we do not know if hypoxanthine is required for TF activity. While it is attractive to speculate that TF contains 1 to 2 nucleic acid bases with a short peptide chain, there is currently no evidence that that indeed is the structure of transfer factor.
VOLUME 58 NUMBER 1. PART 2
Transfer
factor
I
REFERENCES 1 Lawrence, H. S.: Transfer factor, Adv. Immunol. 11: 195, 1969. 2 Kirkpatrick, C. H., and Gallin, J. I.: Treatment of immunologic and infectious diseases with transfer factor, Oncology 29: 46, 1974. 3 Baram, P., Yuan, L., and Mosko, M. M.: Studies on the transfer of human delayed-type sensitivity, J. Immunol. 97: 407, 1966. 4 Neidhart, J. A., Schwartz, R. S., Hurtubise, P. E., Murphy, S. G., Metz, E. N., Balcerznk, S. P., and LoBuglio, A. F. : Transfer factor: Isolation of a biologically active component, Cell. Immunol. 9: 319, 1973. 5 Gallin, J. I., and Kirkpatrick, C. H.: The chemotactic activity in dialyzable transfer factor, Proc. Natl. Acad. Sci. U.S.A. 71: 498, 1974. 6 Gottlieb, A. A.: In Brent, L., and Holborow, J., editors: Progress in immunology, II, Vol. 5, New York, 1974, American Elsevier, p. 375. 7 Mendes, N. T., Tolmai, N. E. A., Silveira, N. P. A., Gilbertson, R,. B., and Metzgar, R. S.: Technical aspects of the rosette tests used to detect human complement receptor (B) and sheep erythrocyte-binding (T) lymphocytes, J. Immunol. 111: 860, 1973. 8 Kirkpatrick, C. H.: Properties and activities of transfer factor, J. ALLERGY CLIS. IXMUNOL. 55: 411, 1975. 9 industrial Method, AI1 93.7OW, January, 1971, Technicon Industrial Systems, Tarrytown, N. Y. 10 Schneider, W. E.: In Colowick, S. P., and Kaplan, N. O., editors: Methods in enzymology, III. New York, 1957, Academic Press, Inc., pp. 99, 680. 11 BShlen, P., Stein, S., Dairman, W., and Udenfriend, S.: Fluorometric assay of proteins in the nanogram range, Arch. Biochem. Biophys. 155: 213, 1973. pp. 734-755, chromatography, New York, 1969, Springer-Verlag, 12 Stahl, E.: Thin-layer 792801. 13 Hopkinson, D. A., Cook, P. J. L., and Harris, H.: Further data on the adenosine deaminasc (ADA) polymorphism and a report of a new phentoype, Ann. Hum. Genet. 32: 361, 1969.
M.D.,
Ruth Knight,
MS.,
and Malvin
Stern,
Transfer factor was prepared from the leu,kocyte lysates of four donors with known skin test reactivity. After ?Lltrafiltration and do&&-gel filtration on polynoylumide gels, fraction IV of the preparation was found to have ‘biologic activity. This fruction contained one major and ocoasionully one minor &raviolet-absorbing a.& zero to on.e ninkydrin-detectable sports on thin-layer chromatography. The major ultraviolet spot was identified as kypoxanthine. Hypoxnnthine was demonstrated to be responsible for the high 260 nm/280 nm ratio of preparations with 6iologi.o activity in viuo. It wus not determined if hypoxanthine is reqzcired for transfer factor activity. 1n addition, an orctiol-negative preparation also had biologic activity.
Human leukocyte lysates contain low molecular weight material which has the ability to transfer to a recipient the delayed skin reactivity of the donor. This material has been called transfer factor (TF) by Lawrence1 and his eel,leagues. The existence, specificity, and small size of transfer factor has been confirmed many times. There is, however, little knowledge about its biochemical composition.ls 2 Baram and associates semipurified a material which appeared to be composed primarily of nucleic acid bases. Kirkpatrick and Galliq2z 5 Neidhart and eolleagueq4 and GottliebF have described preparations which elute as low molecular weight materials on Sephadex columns. In this communication, we describe the use of ultrafiltration and polyacrylamide (P-2) gel filtration to prepare transfer factor and thin-layer chromatography and spectrophotometrie scanning to analyze it. TERIALS AND In vivo testing
METHODS
Because of ethical consideration, normal human volunteers were not used. Therefore, transfer factor preparations were tested on two subjects with immunodeficiencies. Other modalFrom the Department of Pathology? Division of Clinical Pathology, Department of Medicine, Upstate Medical Center, State Umversity of New York. Supported in part by General Research Support Grant RR05402 from the Division of Res#earch R.esources, National Institutes of Health. Presented in part at the meeting of the American Association of Immunologists, Atlantic City, N. J., April, 1975. Received for publication Aug. 15, 1975. Accepted for publication Dec. 1, 1975. Reprint requests to: Dr. Russell H. Tomar, M.D., Upstate Medical Center, State University Hospital, 750 East Adams St., Syracuse, N. Y. 13210. Vol. 58, Wo. 1, Part 8, pp. 190-197
Transfer
VOLUME 58 NUMBER 1, PART 2
TAELE I. Preparation
ik:. EL
of human
iti M M
transfer
2: t:
factor:
42 x42 15x20 30x40 50x50
factor
%
Skin test of donors
22x35 11x18 17x20 10x11
7i “t;’ ci
ii is Y0
‘One-tenth milliliter injected intradermally. t SK-SD 100/25 U/ml (Lederle) . $Can&da 1: 50 (IIollister-Stier ), $PPD 5 TU (Connaught). /jHistoplasmin (Parke, Davis & Co.). ities of therapy had failed in both of these patients, The first had chronic mucocutaneous candidiasis and the second had ataxia telangiectasia. Both had negative skin reactions to 0.1 mi intradermal injections of etreptokinase-streptodornase (100/25 units per ml, Lederle) and Candida (l-50, Hollister-Stier) and a decreased number of circulating lymphocytes dlieh spontaneously formed rosettes with sheep erythrocytes (T cells). Transfer factor was tested in two ways as described by Lawrence.1 In the first-the local assay-materials were adjusted to an absorbance at 260 nm of 0.200 to 0.300, i.e., 5% to 10% of the absorbance in Fraction IV. Aliquots of 0.1 ml were injected intradermxlly along the volar aspects of the forearms along with a buffer control. Tlventy-four hours later, antigens to Fvhich the donor of the transfer factor was sensitive mere injected onto the same areas as previously injected with the transfer factor preparations. Reactions were read at 24 and 48 hr after the introduction of antigen 1)~ two observers one of whom had no knowledge of this protocol. All fractions could be tested at the same time in this way. The second procedure for testing transfer factor-the systemic assay-required adjusting absorbance at 260 nm to 0.800 to 1.00. An nliquot of 0.2 ml was injected intradermally. Twenty-four to 48 hr later, antigens were tested in the contralateral ext,remity. Only a single fraction could be assayed at any one t,ime. Again, skin tests were read at 24 and 48 III. by two observers one of whom was aware of the protocol. In addition, spontaneous erythrocyte rosetting by peripheral lymphcytes was determined before rind after transfer factor injection.7, 8 Preparation
of transfer
factor
h unit of blood was drawi from donors selected for their skin reactivity to SK-SD, Candidn, PPD (Connaught), and histoplasmin (Parke, Davis) (Table I). After the buffy coat containing l-2 x 109 leukocytes TX-as removed in 20 to 50 ml of plasma, it was frozen and thawed 4 to 10 times before ultrafiltration with positive pressure (Amicon X-MSO). The filtrate was then gel filtered through a 50 x 1.5 cm polyacrylamide P-2 (Biogel) column with ammonium carbonate buffer (pH 7.4, 0.001 M) (Fig. 1, a). Flow rate was 30 to 40 ml per hour and 3 to 5 ml was collected in each tube. Absorbances at 260 and 280 nm mere determined. The fractions in the individual peaks were pooled and refiltered through the same column (Fig. 1, B). The refiltered peaks were used for further testing and analysis. Approximately 57’ of the original absorbance units at 260 nm were present in the refiltered Fraction 1V. iochemical
analysis
Phosphates mere determined by converting condensed phosphates to orthophosphates with sulfuric acid and determining the phosphate concentration by the reduction of phosphomolyhdic acid with ascorbic acid. ATP adjusted to an optical density of 0.733 and 1.382 at 260 nm (0.045 and 0.090 mg/ml, respectively) was used for reference.9 Ribose was estimated by the orcinol reaction using adenosine, inosine monophosphate, 2’3’ guanosine monophosphate, and cyclic cytidine as references.10 Absorbance maxima mere determined with a recording scanning spectrophotometer (Beck-
192
J. ALLERGY CLIN.
Tomar, Knight, and Stern
---
IMMUNOL. JULY 1976
260nm 280nm A RECHROMATOGRAPHY M.K.IP:
.4 ------
260 mm 260 mm
.3
Tube #
IO
20
30
40
50
Q FIG. 1. A, The ultrafiltered leukocyte lysate was gel filtered through a polyacrylamide (P-21 column of 50 x 1.5 cm using ammonium carbonate (pH 7.3, 0.001 M) as buffer. Flow rate was 30 to 40 ml/hr; samples of 3 to 5 ml per tube were collected and absorbances at 260 nm and 280 nm determined. The tubes comprising the major peaks were combined and dried from the frozen state. Blue dextran eluted at tube 10. 8, The combined (yophilized material was brought up in the same buffer and refiltered on the same column. Shown is a typical refiltration of Fraction IV. The tubes noted by the black bar were combined and used for further testing. man Model 25). Primary amino acids were detected with fluorescamine using phosphate buffer (pH 8.3, 0.067 M) and 10 mg of fluorescamine in 100 ml dioxane.11 Thin-layer chromatography was done on silica G (F2.54) plates (250 p) with butanol:acetic acid:water 4:l: 1 as solvent and on cellulose plates (100 p) with methanol:hydrochloric acid:water 7:2 :1 as solvent. A closed atmosphere system was used. The ascending front was allowed to develop 100 mm. Forty to 60 ~1 of concentrated material (A260 nm = 0.80 to 3.5) were applied. Ultraviolet absorption (254 nm) for nucleic acid constituents, ninhydrin spray for primary amino acids, peptides and proteins, fluorescamine spray for the same materials, and carbon charring with sulfuric acid were used as methods of detection.12 Standard bases, nucleosides, and nucleotides for scanning and thin-layer chromatography were obtained commercially (Sigma) and adjusted to approximately 100 to 200 pg/ml. Xantbine oxidase (Sigma) was adjusted to 0.12 U/ml in phosphate buffer (pH 7.5, 0.067 M). One-tenth milliliter of this enzyme was added to 0.9 ml of Fraction IV and incubated at 37’ 6.13 Spectrophotometric scanning was done before and during incubation. When the reaction was complete, thin-layer chromatography was performed on the treated material as well as on untreated Fraction IV.
Transfer factor activity was demonstrated to be present in Fraction IV. Delayed reactions, although small, were seen only where Fraction IV had been
VOLUME 58 NUMBER 1. PART 2 TABLE
II. “Local”
Transfer
assay
for transfer Patient
1
Patient
3x4 0
5x5 0
4x5 0
2x2 0
0
0
2x2 6x8 0
0 4x5 0
“One-tenth milliliter of material adjusted to 0.200-0.300 absorbance with antigen at the same site 24 hr later. tIndicates donor of transfer factor.
TABLE III. “Systemic”
assay
2
(TF,wt) (mm induration)
0 0
IV Buffer
1
factor
(mm induration)
II ITT
factor
for transfer
8X8
0
3x3 0
at 260 nm before
challenge
factor Peripheral lymphocyte sheep erythrocyte rosettes
January 21 February 4 March 4 March 18 March 29 April 18 April 22 May 8 June 20 June27 August 12 “Indicates iIndicates
0 II (MF*)
0
i O/O?
III (MF*) o/o
IV (MF*)
o/o
2:
6x8 8x10
8x8 10x10
o/o o/o
67 50
10x10
10x12
8x10 8x10 Normal
32260
IV (ww*)
donor of TF. two observers.
previously injected in the LLlocal” assay in both patients (Table II). In the systemic assay, Fractions II and III failed to convert the recipient from negative to positive ; while two preparations of Fraction IV allowed the previously negative recipient to develop skin tests of at least 6 mm induration. Incidently, the number of peripheral lymphocytes which bind sheep erythrocytes appeared to have increased after one patient had been treated with Fraction IV and perhaps III (Table III). Furthermore, this patient’s ungual moniliasis cleared. Results were similar on both thin-layer chromatography systems used. Therefore, only the silica G system results will be described.’ All four of the crude TF preparations contained 3 to 4 ninhydrin and 2 to 4 ultraviolet-detectable spots. Fraction II from all four subjects demonstrated 2 ultraviolet-absorbing spot,s of approximately the same Rf and a large homogeneous ninhydrin-staining area. All Fraction III preparations showed a ninhydrin-staining spot of similar Rf. All demonstrated a second substance-either ultraviolet absorbing (two samples) or ninhydrin staining (the other two samples). All four of the Fraction IV preparations showed one clearly identifiable ultraviolet-absorbing spot. Three of the preparations also contained a second faint ultraviolet-detectable area and a small pale ninhydrin-staining spot (Fig. 2).
194
Tomar,
Knight,
and
J. ALLERGY CLIN.
Stern
IMMUNOL. JULY 1976
//// m = absorbance at 254nm @$ @&$$= ninhydrin detectable
I III
I II
/ EL
FIG. 2. Thin-layer chromatography was done on silica G (F254) coated plates of 250~ thickness. Butanol:acetic acid:water (4:l :l) was the solvent. Twenty to 60 PI of material with an absorbance at 260 nm of 0.800 to 3.5 was applied. The front was allowed to move 100 mm in a closed atmosphere system. Ultraviolet absorbance and ninhydrin were used as methods of detection. Fractions II, III, and IV from a typical run are shown. Fraction III shows only ninhydrin-detectable material; Fraction IV primarily ultravioletabsorbing materials. TABLE IV. Thin-layer
chromatography Substance
Hypoxanthine Guanosine 2’3’ GMP IMP Uracil TF IV MF TF IV MK
[Silica
G [F 2541;
butanol:acetic
acid:li,0
[4:t:I
j]
Rf
43 33 ii :: 44
Spectrophotometric scans of crude material, &-actions II, III, and IV showed peaks of 252, 250, 290, and 249 to 252, respectively (Fig. 3). Scans of the four Fraction IV preparations were quite similar, peaking at 250 to 252 with a 260,’ 280 ratio of 3.0 to 5.1. A number of different bases, nucleosides, and nucleotides were surveyed by spectrophotometric scanning and by thin-layer chromatography and compared to Fraction IV. By scanning, only guanosine, 2’3’ guanosine monophosphate, hypoxanthine, inosine monophosphate (IMP), and uracil demonstrated maximal absorbance near that of Fraction IV. None, however, was superimposable upon Fraction IV. By thin-layer chromatography, hypoxanthine, but not IMP, migrated in a location similar to Fraction IV (Table IV). Xanthine at similar concentrations was not detectable by ultraviolet lamp. The major ultraviolet spot in Fraction IV was no longer demonstrable after treatment with xanthine oxidase-an enzyme which converts hypoxanthine to xanthine and xanthine to uric acid. This proeedure was repeated in three preparations. Xanthine oxidase treatment also shifted the absorbance maxima of the Fraction IV preparations tested, changing the 260/280 ratios from 3-5/l to l/1-3 (Table V).
Transfer
VOLUME 58 NUMBER 1, PART 2
factor
1
252
nm
230
300
230
360 251
290
FRACTIONIU -I!!!!&
FIG. 3. Scanning was done with a double-beam spectrophotometer Scans of the precolumn pool with transfer factor and Fractions typical preparation are shown.
TABLE V. Transfer
factor
I Wavelength (mm)
TF IV (MF)
293 250 2601280
with I
+x0 (55 min)
Alone
230
“Relative
IV treated
xanthine
oxidase
TF IV (MP) Alone
(Beckman II, III, and
Model 25). IV, from a
(X0)* I
+x0 (75 mm)
Hypoxanthine
z!x
XanthinexUric
acid
::*
ii
100
100
ii.2
z:
200
20 2.00
26 0.73
7 2.00
1;: 0.33
0 12.40
22 1.oo
1:: 0.36
absorbance
units.
Inorganic phosphates were determined on samples adjusted to an absorbance of 0.800 at 260 nm. Fraction II had greater than six parts per million phosphate; Fraction III had no detectable phosphate; and Fraction IV had 0.6 parts per million phosphates. ATP with an absorbance of 260 nm of 0.733 contained 2.4 parts per million phosphates. Buffer contained none, Thus, Fraction IV had approximately one fourth the amount of phosphate as concentrations of ATP giving similar absorbance at 260 nm. The amount of orcinol-positive material in Fraction IV varied greatly, indicating that the amount of ribose in each preparation also varied (Table VI).
19
Tomar,
Knight,
TABLE VI. Transfer
1:: IV
(Az,anm= 0.850)
J. ALLERGY CLIN.
and Stern
factor:
3 +
IMMUNOL. JULY 1976
Summary
2.50 290 250
Numbers in parentheses are number of preparations *ATP of approximately same A, = 2.4 ppm. tOrein assay. $Fluorescamine assay compared to phenylalanine.
6.0 None 0.6 tested.
Fraction IV was analyzed for primary amino acids by using fluorescamine. Compared to a phenylalanine standard, there appeared to be 2 to 3 @g/ml of primary amine-containing materials in preparations with an absorbance at 260 nm of 0.850. The amount of primary amino acid in the Fraction IV samples tested was relatively consistent (Table VI). DISCUSSION Fraction IV of our transfer fraction preparation is similar in many ways to previously described preparations. l-6 It elutes as a small molecular weight material by gel filtration and transfers in a delayed fashion to an intradermal antigenic challenge. Neidhart and associates and Kirkpatrick’s preparations eluted late from Sephadex gel columns and had a high 260/280 ratio and also transferred skin tests. Baram and colleagues’ small molecular weight transfer factor peaked at 258 nm ; Fraction IV peaks at 249 to 252. Baram’s group did not describe paper chromatography before acid hydrolysis; Fraction IV has one major ultraviolet-absorbing spot prior to hydrolysis. After hydrolysis, Baram and assoGates3 found three ultraviolet-absorbing spots which corresponded to adeniae, guanine, and cytidylic acid. They found no ninhydrin-detectable materials and neither did we in several of our preparations. The fluorescamine determination for primary amino acids, peptides and proteins has not been performed on previously described preparations. It is more sensitive than ninhydrin. I1 It is interesting to note that the primary amino acid content of different TF IV preparations was relatively constant while orcinolpositive material varied greatly, Since we do not know the relative activities of our materials, we cannot calculate specific activities on the basis of either amino acid or ribose determinations. The high 260/280 ratio and positive orcinol tests have been the chief reasons for believing that transfer factor contains oligonucleotides. However, it appears as if hypoxanthine and perhaps xanthine-probably as preparative contaminants -give TF its absorbance characteristics. Moreover, our preparations had a variable amount of orcinol-positive material including some with in vivo activity with no detectable ribose. Since we were unable to test our materials after treatment with xanthine oxidase, we do not know if hypoxanthine is required for TF activity. While it is attractive to speculate that TF contains 1 to 2 nucleic acid bases with a short peptide chain, there is currently no evidence that that indeed is the structure of transfer factor.
VOLUME 58 NUMBER 1. PART 2
Transfer
factor
I
REFERENCES 1 Lawrence, H. S.: Transfer factor, Adv. Immunol. 11: 195, 1969. 2 Kirkpatrick, C. H., and Gallin, J. I.: Treatment of immunologic and infectious diseases with transfer factor, Oncology 29: 46, 1974. 3 Baram, P., Yuan, L., and Mosko, M. M.: Studies on the transfer of human delayed-type sensitivity, J. Immunol. 97: 407, 1966. 4 Neidhart, J. A., Schwartz, R. S., Hurtubise, P. E., Murphy, S. G., Metz, E. N., Balcerznk, S. P., and LoBuglio, A. F. : Transfer factor: Isolation of a biologically active component, Cell. Immunol. 9: 319, 1973. 5 Gallin, J. I., and Kirkpatrick, C. H.: The chemotactic activity in dialyzable transfer factor, Proc. Natl. Acad. Sci. U.S.A. 71: 498, 1974. 6 Gottlieb, A. A.: In Brent, L., and Holborow, J., editors: Progress in immunology, II, Vol. 5, New York, 1974, American Elsevier, p. 375. 7 Mendes, N. T., Tolmai, N. E. A., Silveira, N. P. A., Gilbertson, R,. B., and Metzgar, R. S.: Technical aspects of the rosette tests used to detect human complement receptor (B) and sheep erythrocyte-binding (T) lymphocytes, J. Immunol. 111: 860, 1973. 8 Kirkpatrick, C. H.: Properties and activities of transfer factor, J. ALLERGY CLIS. IXMUNOL. 55: 411, 1975. 9 industrial Method, AI1 93.7OW, January, 1971, Technicon Industrial Systems, Tarrytown, N. Y. 10 Schneider, W. E.: In Colowick, S. P., and Kaplan, N. O., editors: Methods in enzymology, III. New York, 1957, Academic Press, Inc., pp. 99, 680. 11 BShlen, P., Stein, S., Dairman, W., and Udenfriend, S.: Fluorometric assay of proteins in the nanogram range, Arch. Biochem. Biophys. 155: 213, 1973. pp. 734-755, chromatography, New York, 1969, Springer-Verlag, 12 Stahl, E.: Thin-layer 792801. 13 Hopkinson, D. A., Cook, P. J. L., and Harris, H.: Further data on the adenosine deaminasc (ADA) polymorphism and a report of a new phentoype, Ann. Hum. Genet. 32: 361, 1969.