Analysis on protein patterns in the human plasma following long-term constantly physical exercise
Anuwat wonthong 1 , Khomsorn Lomthaisong 2 , Wachira Wan 2 , Sompong Thammasiriruk 2
1 Graduate school, Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon
Kaen, 40002
2 Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002
We interested in protein patterns in human plasma following constantly physical exercise for 8 weeks. Five male volunteers with the average age of 24.4

4.04 years and average body mass index (BMI) of 22.10

1.69 were applied in this experiment. 2-dimensional electrophoresis (2-DE) was employed to separate proteins of pre- and post-physical exercise plasma. When using 3-10 pH linear IPG strip, we found that 5 protein spots were increased in the post-physical exercise. Protein spots were identified by compared their pI and MW to known published protein databases and found that spot 1 and spot 2 are haptoglobin protein, while spot 3, 4, and 5 are not identifiable. When 3-10 pH non-linear IPG strip were used, we found that there are clear differences of protein patterns from pre- and post-exercise plasma. We found 4 protein spots were increased their volume and identified as plasma retinal-binding protein (PRBP), haptoglobin and kininogen respectively. Moreover, we found 3 protein spots were decrease in the post-exercise plasma. However, these protein spots are not yet identifiable when compared to known proteins in the databases. In conclusion, constantly physical activity has an influence on particular protein level in plasma. These results could be helpful to account the relationship of such proteins with their presence in the plasma in term of their functions involved in the healthiness following long-term physical exercise.
Introduction
Nowadays, the life styles of Thai people were changes from the past. People worry about their economic status and they need more money to support their life. So, people have to working hard and have no time to take care themselves. The bad behaviors of people for example lack of take a rest, lack of exercise, mistake nutrient, smoking and etc cause of many sickness such as hypertension, heart disease and cancer. In Thailand, the failure of blood circulation system is the first cause of people dead (the Ministry of Public Health of Thailand).
An exercise is important activity and necessary for normal living of people. It can be increase efficiency of many systems in our body such as respiratory system, blood circulation, and immune system. Moreover, an exercise can be also increase strength of body muscle and flexibility of joint and exercise can help a fat guy to loss a body weight too. Numerous researches found that people who have usually exercise were risk to heart disease and hypertension less than people who have no exercise about twenty to forty percents. In the observation of Thailand National Statistical
Office in 1997 found that Thai people were lack of exercise. The PAHO suggest that people should be used time to exercise at least thirty minute per day and three days a week or follow the physical condition of each person.
Much information of protein expression patterns from secretion s including human plasma were kept on protein data base and the two dimension polyacrylamide gel electrophoresis was a principle to use for the study on protein expression patterns.
This technique can isolate a thousand of protein types at the same time. Proteins were separated by its two properties, pI and molecular weight. So, when the exercise is

happened, the plasma proteins may be changes and it may involve in changes of the physical condition of our body. Therefore, we can study the changes of protein expression patterns of human plasma after an exercise and identified the changed proteins by compare with the information on protein data base.
Methodology
Five male volunteers with the average age of 24.4

4.04 years and average body mass index (BMI) of 22.10

1.69 were used in this experiment. Volunteers have no record of exercise over a month. When the experiments start, plasmas were collected and kept at -70 o
C until used. Then, volunteers were continuously exercised by running at least thirty minute per day and three days a week for eight weeks. At the end of exercise, plasmas were collected again. Pre- and post-physical exercise plasmas were used for study together. The impurity of plasma protein was discarded by 2-D Clean-up kit protocol (Amersham Biosciences) and proteins concentration was determined using 2-D Quant kit (Amersham Biosciences) and BSA standards. 2dimensional electrophoresis (2-DE) was used to separate the plasma proteins of pre- and post-physical exercise volunteers. First dimension IEF was carried out using an
Ettan IPGPhor IEF System (Amersham Biosciences) and Immobiline DryStrip gels (7 cm: p I 3-10 L and NL, Amersham Biosciences) were used. Focussed IPG strips were equilibrated for 15 min in equilibration solution (50mM Tris-HCl, pH 8.8, 6M urea,
30% glycerol, 2% SDS, 0.01% bromophenol blue) containing 5 mM dithiothreitol and then alkylated for a further 15 min in equilibration solution containing 13.5 mM iodoacetamide. Electrophoresis was carried out using 7 cm 12.5% SDS-PAGE gels.
Following SDS-PAGE, protein spots were visualized using the PlusOne Silver
Staining Kit (Amersham Biosciences). The protein patterns were analyzed with program ImageMaster
TM
2D Platinum (Trial version).
A B C D
Figure1. The plasma proteins obtained form Pre- (A and C) and post- (B and D) physical exercise volunteers. Protein isolations were performed by two type of IEF separation, linear gradient pH 3-10
IPG strip (A and B) and non- linear gradient pH 3-10 IPG strip (C and D).
Result, conclusion and discussion
The 2DE gels of the plasma proteins obtained form Pre- and post-physical exercise volunteers are shown in Figure 1 and the changed spots are showed in Figure
2. A number of protein spots showed clear differences in intensity that could be revealed even visually. When using 3-10 pH linear IPG strip, we found that 5 protein spots were increased in the post-physical exercise. Protein spots were identified by compared their pI and MW to known published protein databases and found that spot
1 and spot 2 are haptoglobin protein, while spot 3, 4, and 5 are not identifiable. When

3-10 pH non-linear IPG strip were used, we found that there are clear differences of protein patterns from pre- and post-exercise plasma. We found 4 protein spots were increased their volume and identified as plasma retinal-binding protein (PRBP), haptoglobin and kininogen respectively. Moreover, we found 3 protein spots were decrease in the post-exercise plasma. However, these protein spots are not yet identifiable when compared to known proteins in the databases.
5
4
7
5
6
1 2
3
4
3 2
2
1
1 2
1
A
B
Figure2. The changes plasma protein spots obtained form Pre- (A1 and B1) and post- (A2 and B2) physical exercise volunteers.. IEF separation of (A) used linear gradient IPG strip pH 3-10 and IEF separation of (B) used non-linear gradient IPG strip pH 3-10.
Table1. Prediction of rat’s adrenal gland proteins using molecular weight and pI analysis pI range
Spot
No.
1 pI
5.72
MW (kDa)
14
Regulation
Up regulated
Predicted protein haptoglobin
3-10 linear
2
3
6.05
7.77
14
16
Up regulated
Up regulated CD 160 antigen
4
5
1
6.3
6.5
7.12
38
55
12
Up regulated
Up regulated
Up regulated
HLA class I, FK506BP
IMD I, LIRA2 kininogen
5
6
7
2
3
4
6.63
6.2
5.74
6.44
6.73
5.86
15
15
16
30
32
33
Up regulated
Up regulated
Up regulated
Down regulated
Down regulated
Down regulated haptoglobin
3-10 non linear
PRBP
HLA II, CAH5B, ficolin2
-
A2GL, ASGR2
The increasing of several proteins, such as haptoglobin, kininogen and plasma retinal-binding protein (PRBP) are of interest. Haptoglobin combines with free plasma hemoglobin, preventing loss of iron through the kidneys and protecting the kidneys from damage by hemoglobin, while making the hemoglobin accessible to degradative enzymes. Kininogens are inhibitors of thiol proteases. HMW-kininogen plays an important role in blood coagulation by helping to position optimally prekallikrein and factor XI next to factor XII. In addition, HMW-kininogen inhibits the thrombin-and plasmin-induced aggregation of thrombocytes. PRBP delivers

retinol from the liver stores to the peripheral tissues. in plasma, the rbp-retinol complex interacts with transthyretin, this prevents its loss by filtration through the kidney glomeruli.
This preliminary result has indicated that exercise has effect on the level of protein in plasma
.
The changes in these protein levels may be involved in the changes and adaptation of physiological condition of our body following exercise.
References
Borges L., Garcia SF., Gomez RF., Vicario JL. (1997
)
HLA class I allele (HLA-A2) expression defect associated with a mutation in its enhancer B inverted CAT box in two families.
Hum Immunol. Vol.41, 69-73
Chunch TK. (2002
)
Exercise cuts inflammation-related protein in blood. Arteriosclerosis,
Thrombosis, and Vascular Biology. Vol.22, 1869-1876
Jaconi S., Rose K., Hughes GJ., Saurat JH., Siegenthaler G. (1995) Characterization of two posttranslationally processed forms of human serum retinol-binding protein: altered ratios in chronic renal failure. J Lipid Res.Vol.36, 1247-1253
LEIGH A. and NORMAN G.A.(1977) High resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci., Vol. 74 , 5421-5425
Pivarnik JM., Montain SJ., Graves JE. and Pollock ML.(1988) Alterations in plasma volume, electrolytes and protein during incremental exercise at different pedal speeds. Eur J Appl
Physiol Occup Physiol. Vol.57, 103-109
World Health Organization, Cardiovascular disease: prevention and control, Search at 11 December
2004, from http://www.who.int/dietphysicalactivity/publications/facts/cvd/en/