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

Bhat, Gopalakrishna P

BS, Varashree

English

Cognitive Sciences ePrint Archive

Short Report:
A Study on Proteolytic Enzyme Activity in the Erythrocytes of Diabetic Patients
BS Varashree, Assistant Professor,
P Gopalakrishna Bhat, Professor,
Department of Biochemistry, Kasturba Medical College, Madhav Nagar, Manipal- 576104, India.
Address For Correspondence:
BS Varashree,
Assistant Professor,
Department of Biochemistry,
Kasturba Medical College,
Madhav Nagar,
Manipal- 576104, India.
E-mail: varasuhas@yahoo.com
Citation:  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
URL: http://www.ojhas.org/issue36/2010-4-13.htm
Open Access Archives: http://cogprints.org/view/subjects/OJHAS.html and http://openmed.nic.in/view/subjects/ojhas.html
Submitted: Sep 28, 2010; Suggested revision: Oct 4, 2010; Resubmitted: Oct 6, 2010; Accepted Oct 14, 2010; Published: Jan 20, 2011
Abstract:
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
Introduction:
Oxidative stress is a result of either overproduction of reactive 
oxygen radicals or decreased efficiency of inhibitory or scav-
enger 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 es-
sential 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  de-
rangements, 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 qual-
ity and functional integrity of proteins by degradation and re-
placement  of  proteins  damaged  by  oxidation  and  glycation. 
Proteolytic  process  preferentially  degrades  the  oxidatively 
modified proteins. Oxygen radicals and other ROS cause modi-
fication  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 in-
hibitors and promote indiscriminate proteolysis. Oxidant dam-
age to proteins could result in changes in the secondary and ter-
tiary conformation of proteins.  These may lead to changes in 
protein function, chemical fragmentation and increased suscept-
ibility to proteolytic attack.(2) Cell proteins with altered struc-
tures may also arise from post synthetic modifications includ-
ing  nonenzymatic  glycosylation,  spontaneous  deamidation  or 
reaction with free radicals and oxidants.(3) Exposure of normal 
human erythrocytes to oxygen radicals can induce oxidant dam-
age to erythrocyte proteins especially hemoglobin. An enzyme 
system exists in erythrocytes which rapidly degrades the oxid-
atively  damaged  proteins.(4) Proteolytic  enzymes  degrade 
many oxidatively altered proteins, thus preventing the accumu-
lation of altered and damaged proteins in the cell. The erythro-
cytes 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 oxid-
ant damaged hemoglobin in diabetes mellitus when compared 
to normal individuals.
Materials and Methods:
Sample collection: The study group comprised of non-diabetic 
individuals and diabetic patients attending the Kasturba hospit-
al, Manipal. Informed consent from the patients was obtained 
for the study. Patients were selected at random and no distinc-
tion was made between those with insulin dependent or non in-
sulin dependent diabetes. The control group included fifty one 
non  diabetic patients  (mean age=54.47).  The test  group  con-
sisted  of  fifty  three  diabetic  individuals  (mean  age=52.92); 
whose fasting glucose level was more than 126mg%. The dia-
betic 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.  
1
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Online Journal of Health and Allied Sciences
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Volume 9, Issue 4; Oct-Dec 2010
Preparation of oxidatively damaged haemoglobin substrate:
Preparation of the hemolysate: Blood collected from a volun-
teer  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 pre-
pared 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 es-
timation.
Proteolytic activity in human erythrocytes that degrades ox-
idatively damaged hemoglobin: The erythrocyte contains sev-
eral 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 oxid-
ative  stress.  Proteolytic  activity  in  the  cell-free  extracts  was 
measured as follows. Alanine was used as a standard for the es-
timation of amino groups released during proteolytic degrada-
tion of oxidatively damaged hemoglobin. Amino group concen-
trations in the TCA supernatants were calculated from the alan-
ine standard graph. The difference in the amino group concen-
tration 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 statistic-
ally using students unpaired ‘t’ test, Mann Whitney ‘U’ test and 
Karl Pearson correlation test; p=0.001 was considered statistic-
ally significant.
Results and Discussion:
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)
Figure 1: Proteolytic activities in the diabetic erythrocytes 
compared to the control group
Table 1: Proteolytic activities in the diabetic erythrocytes 
compared to the control group
Parameters Mann- Whitney ‘u’ test
‘p’val
ue
Fasting blood sugar (mg %) 8.61 0.001
Proteolytic activity(nmoles/gHb) 6.24 0.001
The increased proteolytic activity suggests an increased ability 
of the erythrocytes to proteolytically degrade oxidant damaged 
hemoglobin in the diabetic patients when compared to hemo-
lysates 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 de-
grade oxidatively damaged haemoglobin. Proteolytic enzymes 
degrade many oxidatively altered proteins preventing the accu-
mulation  of  altered  and damaged  proteins  in  the  cell.  It  has 
been shown that erythrocyte  membrane proteins become sus-
ceptible  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  pro-
teinase family.(10)To know if the proteolytic system could effi-
ciently work as an antioxidant scavenging system, proteolytic 
activity of erythrocytes extracts was estimated using phenylhy-
drazine treated hemoglobin substrate, the enzymes in the eryth-
rocyte  degrade oxidatively  damaged haemoglobin  and simul-
taneously 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 be-
fore incubation gives an indication of endogenous protein dam-
age due to oxidative stress. In the present study, the proteolytic 
activity in the erythrocyte lysates of diabetic patients was signi-
ficantly increased when compared to normals, which correlated 
with the other studies.(6) Hyperglycemia greatly increased pro-
teolytic 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 lys-
ate.(4,11)    
References:
1. Bruch CG, Pierce JD. Oxidative stress in critically 
ill  patients.  American  Journal  of  Critical  Care 
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2. Droge W. Free radicals in the physiological control 
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2
3. Fagan  JM,  Waxman  L,  Goldberg  AL.  Red  blood 
cells contain a pathway for the degradation of oxid-
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or  ubiquitin.  J  of  Biological  Chemistry 
1986;261(13):5705-5713. 
4. Raghothama C, Rao P. Increased proteolysis and ox-
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5. Raghothama  C,  Rao  P.  Degradation  of  glycated 
hemoglobin  by  erythrocytic  proteolytic  enzymes. 
Clinica Chimica Acta 1996;245:201-208
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7. Peterson GL. Determination of total protein.  Meth-
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damaged erythrocyte  membrane proteins.  Biochem 
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9. Fujino T, Watanabe T, Beppu M, Kikugawa K, Yas-
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Biochem Biophys Acta 2000;1478(1):102-112. 
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Purification  of  human  erythrocyte  proteolytic  en-
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member  of  the  multicatalytic  proteinase  family. 
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11. Davies  KJA,  Goldberg  AL.  Proteins  damaged  by 
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tracts. J Biol Chem 1987;262:8227-8234.
3


Characterization of membrane bound serine protease related to degradation of oxidatively damaged erythrocyte membrane proteins.

Degradation of glycated hemoglobin by erythrocytic proteolytic enzymes.

Determination of total protein.

Free radicals in the physiological control of cell function.

Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase.

Increased proteolysis and oxidatively damaged hemoglobin in erythrocyte lysates in diabetes mellitus.

Oxidative stress in critically ill patients.

Proteins damaged by oxygen radicals are rapidly degraded by RBC extracts.

Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family.

Red blood cells contain a pathway for the degradation of oxidant damaged hemoglobin that does not require ATP or ubiquitin.

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide. Free Radical Biol Metab

A Study on Proteolytic Enzyme Activity in the Erythrocytes of Diabetic Patients

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A Study on Proteolytic Enzyme Activity in the Erythrocytes of Diabetic Patients

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