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OJHAS Vol. 9, Issue 4:
(Oct-Dec, 2010) |
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| A Study
on Proteolytic Enzyme Activity in the Erythrocytes of Diabetic
Patients |
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BS Varashree, Assistant Professor,
P Gopalakrishna Bhat, Professor,
Department of Biochemistry, Kasturba Medical College,
Madhav Nagar, Manipal- 576104, India. |
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Address For Correspondence |
Assistant Professor,
Department of Biochemistry,
Kasturba Medical College,
Madhav Nagar,
Manipal- 576104, India.
E-mail:
varasuhas@yahoo.com |
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Varashree BS, Bhat PGK. A Study
on Proteolytic Enzyme Activity in the Erythrocytes of Diabetic
Patients. Online J Health Allied Scs.
2010;9(4):13 |
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Submitted: Sep 28,
2010; Suggested revision: Oct 4, 2010; Resubmitted: Oct 6, 2010;
Accepted Oct 14, 2010; Published: Jan 20, 2011 |
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| Abstract: |
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The present study demonstrates the possibility of increased proteolytic activities in diabetic individuals.
Proteolytic activity was measured by the amount of amino group released
by the erythrocyte lysate of the diabetic individual using phenylhydrazine
treated hemoglobin as substrate. The proteolytic activity in erythrocyte
lysates against oxidatively damaged hemoglobin was significantly increased
in diabetic individuals compared to controls (p<0.001).The result
of this study indicates that in diabetic individuals, proteolytic enzymes degrade many oxidatively
altered proteins preventing the accumulation of altered and damaged
proteins in the cell.
Key Words:
Oxidative stress; Proteolytic activity; Diabetes mellitus
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Oxidative stress is a result
of either overproduction of reactive oxygen radicals or decreased efficiency
of inhibitory or scavenger systems. Under normal physiological conditions,
there is a critical balance in the generation of oxygen free radicals
and antioxidant defense systems used by organisms to deactivate and
protect themselves against free radical toxicity. Oxidants
are balanced by the activities of enzymes and non-enzymes called antioxidants.(1) This vitally important defense system controls the production and
elimination of oxidants and is essential in controlling the damage that
occurs during oxidative stress. However, when the body is overwhelmed
by increased production of oxidative agents and defense against these
agents is decreased, the ensuing damage contributes to cellular derangements,
cell injury, and death. Proteolytic
enzymes are the second line of defense against the free radicals, which
degrade and eliminate the damaged molecules. Cells
maintain the quality and functional integrity of proteins by degradation
and replacement of proteins damaged by oxidation and glycation. Proteolytic process preferentially
degrades the oxidatively modified proteins. Oxygen radicals and other
ROS cause modification of proteins. Although damage to protein substrate
is likely to cause protein degradation, it is possible that oxygen radical
might activate cellular proteases or damage protease inhibitors and
promote indiscriminate proteolysis. Oxidant damage to proteins could
result in changes in the secondary and tertiary conformation of proteins.
These may lead to changes in protein function, chemical fragmentation
and increased susceptibility to proteolytic attack.(2) Cell proteins
with altered structures may also arise from post synthetic modifications
including nonenzymatic glycosylation, spontaneous deamidation or reaction
with free radicals and oxidants.(3) Exposure of normal human erythrocytes
to oxygen radicals can induce oxidant damage to erythrocyte proteins
especially hemoglobin. An enzyme system exists in erythrocytes which
rapidly degrades the oxidatively damaged proteins.(4) Proteolytic
enzymes degrade many oxidatively altered proteins, thus preventing the
accumulation of altered and damaged proteins in the cell. The erythrocytes
in diabetes are ill equipped to handle increased oxidant stress that it faces. Exposure of normal human
erythrocytes to oxygen radicals can damage the erythrocyte proteins
which lead to increased susceptibility to proteolytic attack. Studies
have suggested an increased activity of erythrocyte proteolytic enzymes
in degrading oxidant damaged hemoglobin in diabetes mellitus.(5) The
present study was carried out to assess the activity of erythrocyte
proteolytic enzymes in degrading oxidant damaged hemoglobin in diabetes
mellitus when compared to normal individuals.
Sample collection:
The study group comprised of
non-diabetic individuals and diabetic patients attending the Kasturba
hospital, Manipal. Informed consent from the patients was obtained for
the study. Patients were selected at random and no distinction was made
between those with insulin dependent or non insulin dependent diabetes.
The control group included fifty one non diabetic patients (mean age=54.47).
The test group consisted of fifty three diabetic individuals (mean age=52.92);
whose fasting glucose level was more than 126mg%. The diabetic status
was assessed by estimating the fasting blood sugar (FBS) using glucose
oxidase method. Proteolytic activity by the amount of amino group released
by the erythrocyte lysate of the diabetic individual using phenylhydrazine
treated hemoglobin as substrate.
Preparation of oxidatively
damaged haemoglobin substrate:
Preparation of the hemolysate: Blood collected from a volunteer
was centrifuged at 3000rpm for 8 minutes. The packed cells were lysed
with 1.5 volumes of water. Then centrifuge at 16,000xg for 20 minutes.
The hemolysate was dialyzed against 0.05M tris HCl buffer with 0.1mM
EDTA (pH 8.3)
Preparation of phenylhydrazine
treated hemoglobin (3): Oxidant damage to hemoglobin
was induced by treating it with phenylhydrazine. The phenyl hydrazine-treated
hemoglobin was adjusted to a final concentration of 50 mg/ml and stored
at -20°C. The above oxidatively damaged hemoglobin was used as substrate
for the proteolytic enzymes of the erythrocyte cell-free extracts prepared from
the study populations.
Estimation of proteolytic
activity in human erythrocytes:
Preparation of erythrocyte
cell free extracts (6): The washed human erythrocytes
were lysed in 1.5 volumes of freshly prepared 1mM DL- dithiothreitol
centrifuged at 16,000xg for 20 minutes. The supernatant was dialyzed
against 10 volumes of buffer using a membrane with molecular weight
cut off of 12- 14 kda. The dialysis buffer contained 20mM Na2HPO4
and NaH2PO4 (pH 7.8), 20% v/v glycerol and 0.5
mM DTT. The dialyzed cell free extracts were used for proteolytic activity
estimation.
Proteolytic
activity in human erythrocytes that degrades oxidatively damaged hemoglobin: The erythrocyte contains several
proteolytic enzymes, some of which are known to degrade oxidatively
damaged hemoglobin. In this study, when a sample of erythrocyte lysate
is incubated with phenylhydrazine treated hemoglobin at 37°C, the enzymes
in the erythrocyte degrade oxidatively damaged hemoglobin and simultaneously
any other oxidant damaged protein present in the erythrocyte lysate.
The end products of the degradation are smaller peptides, which are
TCA soluble and can be measured as an increase in the number of free
amino groups using spectrofluorimeter.(7) Estimation of free amino
groups in erythrocyte lysates before incubation gives an indication
of endogenous protein damage due to oxidative stress. Proteolytic activity
in the cell-free extracts was measured as follows. Alanine was used
as a standard for the estimation of amino groups released during proteolytic
degradation of oxidatively damaged hemoglobin. Amino group concentrations
in the TCA supernatants were calculated from the alanine standard graph.
The difference in the amino group concentration before and after incubation
was taken as a measure of proteolytic activity in the cell-free extract
and was expressed as nanomoles per gram of hemoglobin concentration.
Statistical analysis: All values of analyzed parameters
were ex-pressed as mean ± SD. Statistical analysis was performed using
the Statistical Package for Social Sciences (SPSS/PC; SPSS-13, Chicago,
USA). The results were analyzed statistically using students unpaired ‘t’
test, Mann Whitney ‘U’ test and Karl Pearson correlation test; p=0.001
was considered statistically significant.
The proteolytic activity was
determined in the erythrocytes taken from both individuals with diabetes
mellitus (test group) and normal healthy individuals (control group). Erythrocyte
proteolytic activity was higher in cases (167.2±648 nmoles/gHb) than
the controls (27.9±31.7 nmoles/gHb). FBS did not correlate with the
erythrocyte proteolytic activity. The proteolytic activities in the
diabetic erythrocytes were significantly higher when compared to the
control group (p=0.001) (Table 1, Figure 1).The significantly increased proteolytic activity in
the erythrocyte lysates of diabetic patients, when compared to normals, correlated with the other studies.(6)
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Figure 1:
Proteolytic activities in the diabetic erythrocytes compared to
the control group |
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Table 1: Proteolytic
activities in the diabetic erythrocytes compared to the control group |
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Parameters |
Mann-
Whitney ‘u’ test |
‘p’value |
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Fasting blood sugar
(mg %) |
8.61 |
0.001 |
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Proteolytic activity(nmoles/gHb) |
6.24 |
0.001 |
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The increased proteolytic activity suggests an increased
ability of the erythrocytes to proteolytically degrade oxidant damaged
hemoglobin in the diabetic patients when compared to hemolysates from normal
individuals.
Reactive oxygen species are
known to induce damage to the proteins. This leads to the alteration
in the protein structure and function. In order to degrade these altered
proteins the cells are equipped with proteolytic enzymes. The erythrocyte
contains several proteolytic enzymes, some of which are known to degrade
oxidatively damaged haemoglobin. Proteolytic enzymes degrade many oxidatively
altered proteins preventing the accumulation of altered and damaged
proteins in the cell. It has been shown that erythrocyte membrane
proteins become susceptible to degradation by membrane bound serine
protease activity after oxidative modification of the membranes.(8,9) Studies have demonstrated that in human erythrocytes oxidized
hemoglobin is cleaved into peptides by a high molecular mass proteinase
identified as a member of the multicatalytic proteinase family.(10)To
know if the proteolytic system could efficiently work as an antioxidant
scavenging system, proteolytic activity of erythrocytes extracts was
estimated using phenylhydrazine treated hemoglobin substrate,
the enzymes in the erythrocyte degrade oxidatively damaged haemoglobin
and simultaneously any other oxidatively damaged protein present in
the erythrocyte lysate. The end products of the degradation are a number
of smaller peptides that are TCA soluble and can be measured as an increase
in the number of free amino groups. Estimation of free amino groups
in the erythrocyte lysates before incubation gives an indication of
endogenous protein damage due to oxidative stress.
In the present study, the proteolytic activity in the erythrocyte lysates
of diabetic patients was significantly increased when compared to normals,
which correlated with the other studies.(6) Hyperglycemia greatly increased proteolytic activity as shown by the increase in the concentration of
amino groups released. The erythrocytes of diabetic patients are capable
of disposing off extra load of oxidant damaged hemoglobin. Degradation
of oxidatively damaged hemoglobin is done by the proteolytic system
present in the erythrocyte lysate.(4,11)
- Bruch CG,
Pierce JD. Oxidative stress in critically ill patients.
American Journal of Critical Care 2002;11:543-551
- Droge W. Free
radicals in the physiological control of cell function. Physiol Rev
2002;82:48-56.
- Fagan JM, Waxman L, Goldberg
AL. Red blood cells contain a pathway for
the degradation of oxidant damaged hemoglobin that does not require
ATP or ubiquitin. J of Biological Chemistry 1986;261(13):5705-5713.
- Raghothama C,
Rao P. Increased proteolysis and oxidatively damaged hemoglobin in erythrocyte
lysates in diabetes mellitus. Clinica Chimica Acta 1994;225:65-70.
- Raghothama C,
Rao P. Degradation of glycated hemoglobin by erythrocytic proteolytic
enzymes. Clinica Chimica Acta 1996;245:201-208
- Salo DC, Lin SW, Pacifici RE, Davies KJA. Superoxide dismutase is preferentially
degraded by a proteolytic system from red blood cells following oxidative
modification by hydrogen peroxide. Free Radical Biol Metab 1988;5:335-339.
- Peterson GL. Determination
of total protein. Methods in enzymol 1983;91:95-119
- Fujino T, Ishikawa
T, Inoue M, Beppu M, Kikugawa K. Characterization of membrane bound
serine protease related to degradation of oxidatively damaged erythrocyte
membrane proteins. Biochem Biophys Acta 1998;1374(1-2):47-55.
- Fujino T, Watanabe
T, Beppu M, Kikugawa K, Yasuda H. Identification of oxidized protein
hydrolase of human erythrocytes as acylpeptide hydrolase. Biochem Biophys
Acta 2000;1478(1):102-112.
- Sacchetta P, Battista
P, Santarone S, Di Cola D. Purification of human erythrocyte proteolytic
enzyme responsible for degradation of oxidant damaged hemoglobin. Evidence
for identifying as a member of the multicatalytic proteinase family.
Biochim Biophys Acta 1990;1037(3):337-343.
- Davies KJA, Goldberg
AL. Proteins damaged by oxygen radicals are rapidly degraded by RBC extracts.
J Biol Chem 1987;262:8227-8234.
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