Digestion of Nucleic Acids Starts in Stomach
Yu Liu1, †, Yanfang Zhang1, †, Ping Dong1, *, Ran An1, Changhu Xue1, Yinlin Ge2,
Liangzhou Wei2 & Xingguo Liang1, *
Supplementary Information
1
College of food science and engineering, Ocean University of China, Qingdao 266003, China.
Department of Biochemistry and Molecular Biology, Qingdao University Medical College,
Qingdao 266003, China. † These authors contributed equally to this work. * Present address:
College of food science and engineering, Ocean University of China, Qingdao, 266003, P.R.China.
Correspondence and requests for materials should be addressed to P.D. (dongping@ouc.edu.cn)
or X.L. (liangxg@ouc.edu.cn).
2
Supplementary Figures
Pepsin ＋
a
Time / h
2 24 2 24 2 24 2 24 2 24 2 24 2 24 2 24
2.2
pH value
Lane No. L 1
2.8
2
3 4
3.5
6.0
8.0
5.3
7.0
9 10 11 12 13 14 15 16
4.8
6
5
7 8
10 kb
3 kb
1 kb
250 bp
Pepsin －
b
Time / h
2 24 2 24 2 24 2 24 2 24 2 24 2 24 2 24
pH value
Lane No. L
2.2
1
2
2.8
3 4
3.5
5
6
4.8
7
8
5.3
7.0
6.0
8.0
9 10 11 12 13 14 15 16
10 kb
3 kb
1 kb
250 bp
Supplementary Figure 1: Effect of pH on digestion of  DNA by pepsin. a, Pepsin
(4.0 mg ml-1) was present. b, Pepsin was absent. Significant digestion of DNA was
found when pH < 5.3 (Lanes 1-8, a). Compared with digestion by pepsin (a), the low
pH (acidic condition) itself had almost no effect on digestion (b). Although breakage
of DNA was found after 24 h in the absence of pepsin at pH 2.2 (Lane 2, b), the
effect of pepsin on digestion was significant, especially within 2 h (comparison of
Lane 1 in a with Lane 1 in b). The bands at pH 2.0 was light because the depurination
was carried out at such a low pH and the depurination products were hard to be
stained.
Pepsin ＋
a
2.2 2.8 3.5 4.8 5.3 6.0 7.0 8.0
pH value
Lane No. L
O
1
2
3
4
5
6
7
8
10 kb
3 kb
1 kb
500 bp
Pepsin －
b
pH value
Lane No. L
O
2.2 2.8 3.5 4.8 5.3 6.0 7.0 8.0
8
6
4
5
7
1
2
3
10 kb
3kb
1 kb
500 bp
Supplementary Figure 2: Effect of pH on digestion of salmon sperm DNA by
pepsin. a, Pepsin (4.0 mg ml-1) was present. b, Pepsin was absent. The reaction time
was 2.0 h. For salmon sperm DNA, similar digestion results were obtained as those of
DNA. Phosphate buffered saline with pH higher than 2.2 did not show any effect on
the digestion of salmon sperm DNA (Lanes 2-7, b). However, when 4.0 mg ml-1
pepsin was added, significant digestion of NA was observed after 2 h (Lanes 2-7, a).
a
pH 8.0 pre-treated
Lane No. L
10 kb
＋
－
O 1
2
b
pH 8.0 pre-treated
Lane No. L
N0. 1 N0. 2
－＋ － ＋
O 1 2 3 4
10 kb
3 kb
3 kb
1 kb
1 kb
500 bp
250 bp
Supplementary Figure 3: Inhibition effect of alkaline treatment of pepsin (a) and
gastric juice (b) on salmon sperm DNA digestion. 0.8% agarose gel was used for
electrophoresis. In (a), the alkaline pretreated or non-pretreated pepsin was mixed
with buffer (25 mM NaH2PO4, 200 mM NaCl) and the final pH was 3.8, and reaction
time was 2 h. In (b), digestion was carried out at pH 3.8 for 3 h. Obviously, the
digestion activity was almost lost after alkaline pretreatment for both pepsin (a) and
gastric juice (b).
pH value
Lane No.
3.5 5.3 7.0 8.0
L
O
1
2
3
4
10 kb
3 kb
1 kb
500 bp
Supplementary Figure 4: Effect of pH on digestion of  DNA by DNase I.
Digestion was performed with 1 U DNase I for 0.5 h, other conditions were the same
as those in Fig. 2a. The optimum pH of DNase I on  DNA was 7.0. DNase I was not
inactivated at pH 8.0.
pH value
Lane No.
3.5 5.3 6.0 7.0
L
O
1
2
3
4
10 kb
3 kb
1 kb
500 bp
Supplementary Figure 5: Effect of pH on the digestion of  DNA by DNase II.
Digestion was performed with 0.04 mg ml-1 of DNase II, and other conditions were
the same as those in Fig. 2a. DNase II showed activity even when pH was 7.0 (Lane
4). This was different from the case of pepsin, in which pepsin lost its activity when
pH was higher than 6.0.
a
b
KDa
mV
1200
1000
180
800
75
M pepsin
600
400
48
200
-200 0
34.6 KDa
35
0
5
c
10
Time / min
20
15
25
17
1200
1000
mV
800
600
d
400
200
KDa
0
-200 0
5
e
10
Time / min
15
20
M
rp
mp
180
75
48
35
1400
37.6 KDa
25
mV
1200
1000
17
800
11
600
400
200
0
-200 0
5
10
Time / min
15
20
Supplementary Figure 6: Determination of the purity of commercially provided,
recombinant and mutant pepsin. a, HPLC analysis of commercially provided
pepsin. b, SDS-PAGE analysis of commercially provided pepsin. c, HPLC analysis of
recombinant pepsin. d, SDS-PAGE analysis of recombinant and mutant pepsin. e,
HPLC analysis of mutant pepsin. HPLC conditions: TSKgel G2000SWxl, UV 280 nm;
flow rate, 0.5 ml min-1; mobile phase, 25 mM Na2HPO4-NaH2PO4 buffer (pH 6.0,
containing 25 mM NaCl). Purity of commercially provided pepsin was determined as
98.6% (a). The purity for both recombinant pepsin and mutant pepsin was determined
to be higher than 98%. The SDS-PAGE analysis showed that the molecular weight of
mutant pepsin was 37.6 KDa, which was consistent with the calculated one (d).
a
b
Time / h
0 0.5 1
Lane No. L O 1 2
2
5 12 24
3
4
Time / h
0 0.5 1 1.5 2 3
Lane No. L O 1 2 3 4 5
5 6
5 12 24
6
7 8
10 kb
10 kb
3 kb
3 kb
1 kb
1 kb
500 bp
500 bp
Supplementary Figure 7: Time course of  DNA or salmon sperm DNA digestion
by pepsin. a,  DNA. b, Salmon sperm DNA. The reaction time was 0.5 h, 1 h, 1.5 h,
2 h, 5 h, 12 h and 24 h. Other conditions: 4.0 mg ml-1pepsin, NaH2PO4 buffer (pH 3.8,
200 mM NaCl), 37 °C. The digestion products of  DNA (a) were all oligonucleotides
with the length from several dozens to several hundred of nucleotides, while
mononucleotide was not found even extending the reaction time to 24 h. For salmon
sperm DNA (b), the results were similar to those of  DNA, digestion products were
oligonucleotides rather than mononucleotide.
NaCl Conc. / mM
Lane No. L
0
0
1
40 80 120 150 200 240 280 320 350 400 520
8 9 10 11 12
2 3 4 5 6 7
10 kb
3 kb
1 kb
500 bp
Supplementary Figure 8: Effect of NaCl on the digestion of plasmid pET-28a. With
the increase of NaCl, digestion of plasmid pET-28a slowed down, especially when the
concentration of NaCl was higher than 320 mM (Lane 9). Moreover, almost no
digestion occurred when NaCl was higher than 400 mM (Lane 11).
Time / h
2
Pepsin Conc. (mg / mL)
Lane
No.
24
2 24
0.1
1.0
L
O 1
2
3
4
2 24
5
2
24
2
10-3
10-2
6
7
8
24
10-4
9
10
10 kb
3 kb
1 kb
500 bp
Supplementary Figure 9: Effect of pepsin concentration on digestion of  DNA.
Pepsin concentrations were from 1.0 mg ml-1 to 0.1 μg ml-1. Digestion rate of  DNA
depended greatly on the concentration of pepsin. For 0.1 mg ml-1 of pepsin, the
digestion slowed down, and only tiny digestion was observed within 2 h. However,
the final product still showed an average length of about 300 bp after 24 h of digestion
(Lane 4). The digestion was even observed after 24 h when pepsin concentration was
as low as 1.0 μg ml-1 (Lane 8). When the concentration of pepsin decreased to 0.1 μg
ml-1, no digestion was observed. The pH of digestion solution was 3.8.
16000:1
100:1
Lane No. L O
C 1
2
3
4
5
10 kb
3 kb
1 kb
250 bp
Supplementary Figure 10: Effect of hemoglobin on digestion of  DNA. Lane L,
DNA ladder; Lane O, original  DNA; Lane C,  DNA and Hb (100:1, M/M), a
control in the absence of pepsin; Lane 1,  DNA hydrolyzed by pepsin without Hb;
Lanes 2 to 5,  DNA and Hb (Hb :  DNA = 100:1, 1,000:1, 10,000:1 and 16,000:1;
M/M) hydrolyzed by pepsin. Comparing Lane 1 (without Hb) with Lanes 2-5 (with
Hb), we concluded that NAs could be digested efficiently by pepsin in the presence of
Hb, and protein did not show inhibition effect on NA digestion. The result
strengthened our suggestion that NAs could or tended to be digested by pepsin even
ingested together with protein.
a
1.2
1.0
Digested DNA / %
0.8
0.6
0.4
0.2
0.0
0
50
100
(0.2)
b
150
200
250
Time/min
700
600
y = 1584.5x + 28.506
R² = 0.9979
1/v
500
400
300
200
100
0
-0.1
0
0.1
-100
0.2
0.3
0.4
1/[E]
c
[E] (μmol L -1)
1/[E]
V0 (μmol -1 min -1)
1/v
115
0.00870
0.029
34.48
28.5
0.0351
0.0116
86.21
17.1
0.0585
0.0074
135.14
11.5
5.7
0.0870
0.1754
0.0060
166.67
0.0034
294.12
2.85
0.3509
0.0017
588.24
Supplementary Figure 11: Kinetic analysis of pepsin using S82 as the substrate. a,
Time course of a reaction in the case of 115 μM pepsin. b, Determination of Km and
Vmax for S82 using the Line weaver-Burk plot, the kinetic parameters were calculated
as: Vmax = 0.035 μM-1 min-1, Km = 55.6 μM, kcat/Km =1.05×10-2 s-1 mM-1. The data for
drawing weaver-Burk plot (b) were listed in c.
Supplementary Table 1: Primers for cloning and mutating porcine pepsinogen
Primer name
Sequence (5-3)
32F1
TGGAGATTGGGAGCCAGGAAAGAAC
32R1
GGTTGGAGGAGCCGGTGGCAAAGATGACGGTGAAG
32F2
CTTCACCGTCATCTTTGCCACCGGCTCCTCCAACC
32R2
GCGGGGATAGAACCAAGGCGGGAT
215F1
TGGAGATTGGGAGCCAGGAAAGAAC
215R1
GCAGAGAGGTGCCCGTAGCCACAATGGCCTGGCAG
215F2
CTGCCAGGCCATTGTGGCTACGGGCACCTCTCTGC
215R2
CACGATCCCAGAGGAAAAGCGATCAGA
PF
CTTGAATTCAAAGAACCATGAAGTGGCTAC
PR
TATGCGGCCGCGATAGAACCAAGGCG
5AOX1
GACTGGTTCCAATTGACAAGC
3AOX1
GCAAATGGCATTCTGACATCC
Supplementary Material and Methods
Hydrolysis of DNA by DNase I or DNase II. DNase I was purchased from
Fermentas (EN0521) and was stored in buffer solution (pH 7.5, 50 mM Tris-acetate, 1
mM CaCl2 and 50% glycerol). DNase II was purchased from Sigma-Aldrich (D4138)
and was dissolved in water to 4 mg ml-1. Enzyme/NAs/digestion buffer/H2O were
mixed at the ratio of 2:3:5:10 (v/v/v/v) for DNase II or 1:3:5:11 (v/v/v/v) for DNase I.
Other digestion and analysis conditions were the same as those for pepsin digestion.
Hydrolysis of nucleic acids together with Hb. Hemoglobin (Hb) stock solution was
prepared to contain 40 mg ml-1 Hb (Sigma-Aldrich Co., Ltd.) in 65 mM HCl. Hb
stock solution was diluted by 65 mM HCl to 155 μM, and then diluted to 95.1 μM,
9.51 μM and 0.951 μM by water. Then they were mixed with the same volume of
DNA (300 μg ml-1, 9.51 nM) to produce a series of mixture Hb and DNA. Ratios
of Hb/DNA were 10,000:1, 1,000:1 and 100:1 (M/M). To get higher concentration
of Hb (Hb/DNA=16,000:1), we mixed the same volume of Hb solution (155 μM)
and DNA (9.51 nM). Then 10 μl of digestion buffer (pH 3.0, 200 mM NaCl), 6 μl of
H2O and 4 μl of pepsin stock solution were added. Digestion and electrophoresis were
then carried out as described in “methods” section.