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Polyacrylamide Gel Electrophoresis of Human Salivary Histidine-

Dental Sciences Department, Naval Medical Research Institute, Bethesda
Maryland 20014, USA

A method of polyacrylamide gel electro- nique to allow for excellent resolution of HRP
phoresis for examining histidine-rich-polypep- fractions. The purpose of this report is to de-
tides in human saliva is described. Comparison scribe these conditions.
is made to several commonly used electrophor-
retic techniques. The described method allows Materials and Methods
for the resolution of seven histidine-rich-poly-
peptide fractions and is convenient and quite SAMPLE COLLECTION AND PREPARATION.-
reproducible. For these experiments saliva was obtained from
nine individuals (six male, three female).
J Dent Res 56(9): 1115-1118 September 1977. Parotid saliva was collected using a modified
Carlson-Crittenden cup with sour lemon drop
stimulation. Whole saliva was collected by hav-
Electrophoretic techniques have been widely ing a subject expectorate into a vial while chew-
used for analysis of the protein components of ing paraffin. The whole saliva was centrifuged
human saliva.1 It is well understood that no one at 6,000 g for ten minutes and the resulting su-
electrophoretic method provides adequate in- pernatant used for these studies. Fresh samples
formation on all salivary proteins. (100 gl) of saliva were usually used for electro-
Recently several laboratories have been phoresis. At other times, saliva was lyophilized
interested in a group of histidine-rich-polypep- immediately and then reconstituted to its orig-
tides (HRP) found in human parotid saliva.2-5 inal volume using 8 M urea buffered in 0.37 M
The IURP have certain biological and chemical glycine, pH 4.0. HRP in samples dissolved in
properties which render analysis by common buffered urea migrate more cathodal than HRP
electrophoretic techniques deceptive. The HRP in fresh saliva samples.
are small molecules (3,000 to 6,000 daltons)
related by a degradative phenomenon.2 The ap- Various isolated HRP fractions were also
parent progenitor molecule (termed HRP-1) examined. These were prepared as described
has a neutral isoelectric point and gives rise to previously.3 For most electrophoretic methods,
the other HRP by a progressive breakdown. isolated lyophilized HRP were dissolved in buf-
These include several extremely cationic frac- fered 8 M turea. All chemicals used were of the
tions (pl > 10).2 highest grade commercially available.
We have observed that the frequently used ELECTROPHORETIC METHODS.- For all
anionic (Davis6) and cationic (Reisfeld et al.7) methods gels were cast to a height of 9 cm in
methods of polyacrylamide gel electrophoresis 0.5 X 12 cm glass tubes. No stacking gels were
were not suitable for simultaneous examination used. All samples were heated at 60 C for two
of all HRP. We have modified another tech- mintutes prior to electrophoresis.
(1) Very cationic.-This is modified from
This work was supported by Navy Medical Research
a description in the Buchler electrophoresis
and Development Command, Research Task MRO41.20.- manual of the pH 2.7 cationic gels.
02.0432. Solution A 60.0 gm acrylamide
Received for publication June 29, 1976. 0.8 gm N,N'-methylenebisacry-
Accepted for publication October 29, 1976.
The assertions and opinions contained herein are lamide
those of the authors and are not to be construed as Solution B 0.24 ml N,N,N',N'-Tetrameth-
reflecting the views of the Navy Department or the
naval service at large. ylethylenediamine
* To whom all correspondence should be addressed:
National Institutes of Health, Building 10, Room 6N260, 50 ml glacial acetic acid
Bethesda, Maryland 20014. 24ml 1MKOH
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1116 BAUM, BIRD & LONGTON J Dent Res September 1977
Additional auiounrits of 1M Results
KOII or (flacial aectic acidl
were added to g-ve the de-
The very c ationic system w:ith gels 15 % int
sired pll (2.41, 2.50, 2.74. amcrxlouide affords the greatest resolutioniof
JIRP fractions (Figure). Optimal results were
3.02) found when the pH of solution B was 2.74 (gel
Solutioni C 120 lug Ammoniurn persulfate A). Seven lec trophoretic bands are seen. When
2 mg Riboflavin the pH of solution B wsas 2.41 or 2.59, six HRP
All oomponents were per 100-ml water. A Cor- bands were seen, and at pH 3.02, five bands
ningr 112 digital pH meter equlipped with a were observed (not shown). Under these con-
combination electrode (#476050) sas used for
all plI determinations. Gels were formed by
ditions of electrophoresis, lysozymie migrates
mixing I part solution A: 2 parts solution B: less cathodal than all HRP fractions. When
part soliution C and photopoly nerived for about
20 minutes at room temnperatuire. The upper
tray buffer 0.37 M glycine adjusted to pl"I

4.0 with glacial acetic acid. The lower buffer

was 0.72 M acetic acid adjuisted to pH 4.3
with IM KOH. Methyl green xsas used as a
track dve. A current of 1 mA/ tube wsas used
for 30 usinistes and then increased to 2.5 mA/
tube until electrophoresis wxas completed (dye
within I cm of gel bottomi).
(2) Cationic (Rcisfeld, Lewzs and Wil-
liams7).- The imethod xwas used at described.
No track dve wxas used. Cturrent and time of
electrophoresis were varied. Details are given
with figure legend.
(3) C(ationic with urea (PanVim and
Chalkly8)%.-The method was used as de-
scribed. Samples were dissolved in 0.9 M acetic
acid, 15% sucrose. Pxrsnin GS wavs tised as a
tlacmk dye. As su!ogested by these .muthorss. gels
xs-ver placed in an ice bath for 15 minutes to
fat ilitate remoxal. 'Ilhis also dotloe in tlb

v rv ca tionic s steml.
(41) 4nionic (Davisi). The mllethiod xsas
used as descril)ed. Samples wxxere ruin in either
bufTered 8 M4 urea or 1O% sucrose. Separate
experimiients showed resuilts ivitlh eithes solvent
xvere idenltical. Bromphenol blue was uised as a
tras k dxvc. Gels we-ve ellc troplhoresecd at 1 mnA/
tube for 30 minuites, then 3 mA/tube until FiG.-Suimmary of eleetrophoretic results.
c omplete.
A, fresh parotid saliva subjected to electrophor-
esis on- xvcry cationic sv stens., 1.5 c. acrylanside,
!)Sodin Dodecyl Sdlfac (S1DS) (Fnr- solution B ot pH- 2.74, cathode at bottom. B,
tlssunvsr'i d Tiins t9) The usetlhod Nx as used fresh parotid saliva subjected to electrophoresis
ats descrilbed. S-imples Nwere dissoxlved in 0.01f) in the system of Reisfeld et al.7 Gel is lYe,"
l)losl)hate buffet, pl-I 7.2, containiiin 0.2% SDS aerylautide with the cathiode at the buttons. Elec-
asId 2 MsI cirea, witl anid witlhout I c3-metcap- trol)horesis was fur 1 inA\.tube for 30 nsiissutes,
loethanol. then 8 nmA/tub)e for 75 minutes. C, clectrophor-
Gels riim wxithl SDS prseset xsere stainci esis of pooled H-RP 1-7 in SDS with gel 7.5%/
in 0.51; Cooimissie blue in 20O ttrichloroacetic acrylanilde. Xniode at hottoro1. D, elec trophoresis
acid for onie h1our and diffusion destained of mixtuire of J-IRP 3-7 with trace of JIRP-l,
against 10% ssethanol-7% acetic acid. Other 2 in anionic system. Gel is 7.5tc,- acrylamide
with the anode at the bottorn. E, miixtuire of
gels xv ere stained for one hoLur in 0.5% amiclo
HRP 1-7 subjected to electrophoresis in the
blasck its 7 acetic acid anId diffusioss destained satiouie systens withi urea. GTel is 15St' acryla-
against 7% acetic acid. mniide and cathode is at the bottom.

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electrophoresis is performed in gels of 7.5% acid pH, showed good resolution of protein
acrylamide, there is no resolution of individual components in human parotid and whole sa-
HRP fractions, all seven bands being coalesced livas. Several components, probably HRP, are
(not shown). well resolved. Since isolated, characterized frac-
When the method of Reisfeld et al.7 was tions ere not examined, it is difficult to make
used, results such as those found in gel B were exact comparisons between methods.
observed. HRP-1 and 2 are wvell resolved, how- Azen has done genetic and phylogenetic
ever, there is inadequate resolution of HRP 3 studies on HRP using electrophoretic tech-
and 4 and HRP 5-7. Under these conditions niques.5"'1 His method, an acid-urea-starch gel,
lysozyme migrates just cathodal to HRP-l' (not offers good resolution, but is somewhat cumber-
shown). Electrophoresis with gels 7.5% in acry- some and more time consuming than the poly-
lamide does not improve HRP resolution. Gel acrylamide technique used here. Also, we have
C represents electrophoresis of pooled HRP not found it necessary to concentrate samples
1-7 in the presence of the anionic detergent as he did, finding fresh salivas adequate.
SDS with 7.5% acrylamide. There is no reso- Many investigators have examined saliva
lution of the various fractions under these con- using the procedure of Reisfeld et al.7 As we
ditions, with or without 13-mercaptoethanol have demonstrated, resolution of HRP is sub-
present. With gels 10% in acrylamide results optimal with this method. The use of anionic
were identical. conditions (Davis6) is only adequsate to exam-
When the method of Davis6 was used with ine HRP 1 and 2. Additional HRP (degraded
gels either 7.5% or 15% in acrylamide only fractions) do not penetrate the gel. If break-
HRP-1 and 2 penetrate the gel (D). Here a down were to occur in isolated fractions or sa-
mixture of HRP 3-7, containing trace amounts liva samples, it would go undetected with this
of HRP-1 and 2, was subjected to electro- method. The technique of Panyim and Chalk-
phoresis. The primary migration positions of ley8 and the use of SDS in electrophoresis9 were
HRP-l and 2 are indicated. HRP 3-7 do not likewise found inadequate.
enter the gel at these pH conditions. A similar
mixture of all HRP fractions was examinied by Conclusions
the method of Panyim and Chalkey8 (gel E).
No resolution of the fractions occurred with Most common gel electrophoretic tech-
only one cathodal band resulting. niques are inadequiate for examining the histi-
dine-rich-polypeptides in human saliva. The
Discussion very cationic method described here allows for
the resolution of seven components in this group,
We have described a method of polyacry- including an apparent neutral progenitor mole-
lamide gel electrophoresis useful for examining cule and its cationic breakdown products. The
histidine-rich-polypeptides found in human sa- method is useful for monitoring the isolation of
liva. The method is uncomplicated and quite HRP fractions, studying the process of IIRP
reproducible. As demonstrated above, resolu- degradation, and genetic and phylogenetic stud-
tion of these fractions is sensitive to gel pH and ies of these molecules.
acrylamide concentration. Under optimal con-
The authors thank Dr. G. Clark for helpful com-
ditions in the very cationic system, seven elec- ments on the manuscript; Mrs. N. M. Cox for prepara-
trophoretic bands, corresponding to HRP frac- tion of the manuscript, and the NMRI photography
tions, are visualized. Previously we reported five laboratory for photographic assistance.
HRP fractions in parotid saliva visible on poly-
acrylamide gel electrophoresis and termed References
these HRP 1-5.2 As shown here, HRP-5 resolves
into three components now termed 5-7. We 1. MANDEL, I.D.: Electrophoretic Studies of
have also noted2 that whole saliva contained Saliva, J Dent Res 45:634-643, 1966.
predominantly HRP-5. When whole saliva is 2. BAUM, B.J.; BIRD, J.L.; MILLAR, D.B.; and
subjected to electrophoresis as above, all but a LONGTON, R.W.: Studies on Histidine-Rich-
Polypeptides from Human Parotid Saliva,
trace of HRP material is found as HRP-7 (not Arch Biochem Biophys 177:427-436, 1976.
shown). 3. HAY, D.I.: Fractionation of Human Saliv-
Several other workers have examined ary Proteins and the Isolation of an Histi-
"basic" molecules (the histidine-rich-polypep- dine-Rich-Acidic Peptide Which Shows
tides) by electrophoretic techniques. Boiiilla High Affinity for Hydroxyapatite Surfaces,
and Stringham.l' using 14% cyanogum gels at Arch Oral Biol 20:553-558, 1975.

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1118 BAUM, BIRD & LONGTON J Dent Res September 1977
4. HOLBROOK, I.B., and MOLAN, P.C.: The 8. PANYIM, S., and CHALKEY, A.: High Res-
Identification of a Peptide in Human Paro- olution Acrylamide Gel Electrophoresis of
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the Glycolytic Activity of Salivary Micro- 346, 1969.
organisms, Biochem 1 119:489-492, 1975. 9. FURTHMAYR, H., and TIMPL, R.: Charac-
5. AZEN, E.A.: Genetic Polymorphism of Basic terization of Collagen Peptides by Sodium
Proteins from Parotid Saliva, Science 176: Dodecylsulfate-Polyacrylamide Electrophor-
673-674, 1972. esis, Anal Biochem 41:510-516, 1971.
6. DAVIS, B.J.: Disc Electrophoresis. II. Method 10. BONILLA, C.A., and STRINGHAM, JR., R.M.:
and Application to Human Serum Proteins, Electrophoresis of Human Salivary Secre-
Ann NY Acad Sci 121:404-427, 1964. tions at Acid pH, J Chromatog 50:345-348,
7. REISFELD, R.A.; LEWIs, U.J.; and WIL- 1970.
LIAMS, D.E.: Disk Electrophoresis of Basic 11. AZEN, E.A.: Properties of Salivary Basic
Proteins and Peptides on Polyacrylamide Proteins Showing Polymorphism, Biochem
Gels, Nature 195:281-283, 1962. Genet 9:69-86, 1973.

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