PURIFICATION AND C HARACTERIZATION OF PRO TEASES FROM … · a farm on the Brits Road (Sample 2) and a village in the North West Province (Sample 3). The sam-ples were collected using

PURIFICATION AND CHARACTERIZATION OF PROTEASESFROM COW DUNG: VALIDATION OF AN ANCIENT AFRICANHOME TREATMENT FOR BURN WOUNDS

PURIFICATION ET CARACTÉRISATION DES PROTÉASES DE BOUSESDE VACHE: VALIDATION D’UN VIEUX TRAITEMENT EMPIRIQUE AFRI-CAIN DES BRÛLURES

Gololo S.S., Makhubela S.D., Tshidino T., Mogale M.A.

Sefako Makgatho Health Sciences University, Pretoria, South Africa

SUMMARY. Mankind has for many years relied on home remedies to manage ailments and injuries, includingburn wounds. Wound healing is generally regarded as a natural process, with interventions such as ointment ap-plication regarded as assisting the process. Amongst other strategies, cow dung is a home remedy used by manyAfrican communities to manage burn wounds. The current study was aimed at the detection, purification andcharacterization of proteases from cow dung as validation for its usage in the management of burn wounds amongstAfrican communities. Cow dung was collected around Pretoria, South Africa, and homogenized with a blenderin a phosphate buffer, pH 7. The crude protein was obtained using ammonium sulphate precipitation and purifiedusing size exclusion, anion exchange and cation exchange chromatographic techniques. Enzyme activity was de-termined using the casein digestion method. The purified proteases were characterized according to temperatureand pH as well as substrate specificity. Three protease fractions were purified from cow dung homogenate. FractionH was found to have an optimum temperature of 40˚C, an optimum pH of 6 and affinity for elastin; Fraction Iwas found to have an optimum temperature of 35˚C, an optimum pH of 7 and affinity for haemoglobin; and Frac-tion J was found to have an optimum temperature of 35˚C, an optimum pH of 8 and affinity for collagen. Thefindings of the study therefore suggest that the presence of proteases in cow dung could be one of the contributingfactors towards its effectiveness in traditional African burn wound management.

Keywords: cow dung, proteases, burn wound, chromatography, temperature, pH, substrate specificity

RÉSUMÉ. Les humains ont longtemps utilisé des « recettes de Grand-mère » pour traiter bobos comme blessures,y compris les brûlures. La cicatrisation est considérée comme un phénomène naturel, que divers onguents sontsupposés aider. La bouse de vache est utilisée dans ce but dans de nombreuses communautés africaines. Nousavons détecté, purifié et caractérisé les protéases de bouses effectivement utilisées aux alentours de Pretoria(Afrique du Sud) après homogénéisation à pH 7. Les diverses protéines étaient obtenues par précipitation au sul-fate d’ammonium ; purifiées par exclusions de taille et chromatographies sur résines cationiques comme anio-niques. L’activité enzymatique était déterminée par la digestion de la caséine. Les protéases étaient caractériséesselon les substrats et les pH et températures spécifiques d’activité. Trois protéases ont ainsi été identifiées. Lafraction H est une élastase à température optimale 40°C, pH 6. La fraction I est une hémoglobinase, 35°C, pH7. La fraction J est une collagénase, 35°C, pH 8. La présence de protéases dans la bouse de vache peut expliquersont efficacité en médecine traditionnelle africaine.

Mots-clés : bouse de vache, protéases, brûlure, chromatographie, température, pH, substrat spécifique___________

Corresponding author: Sechene Stanley Gololo, PO Box 235, Medunsa 0204, South Africa. Tel.: +27 12 521 3242; fax: +27 12 521 9806; email: [email protected]: submitted 27/05/2019, accepted 08/06/2019

Annals of Burns and Fire Disasters - vol. XXXII - n. 2 - June 2019

103


104

Introduction

Most African communities have for many years

continuously relied on traditional home remedies to

manage different injury inflicted wounds. This re-

liance on home remedies is mostly driven by finan-

cial constraints that impair access to modern

medical treatments, and is therefore mostly con-

fined to rural areas.1 For the treatment of burn

wounds in particular, a variety of remedies that in-

clude plant extracts, stones, animal fats, oils and an-

imal excretory products such as cow dung and urine

are frequently used.2,3 However, the efficacy of

many of these remedies or intervention agents has

not been validated. Furthermore, information re-

garding the mechanism of action of such remedies

remains scanty.

Amongst the many home remedies for burn

wounds, cow dung is the most commonly used by

many African rural communities.4,3 Like in the case

of many other home remedies, the constituents of

cow dung that contribute to its effectiveness or pre-

sumed efficacy in the treatment or management of

burn wounds are not known. Cow dung is reported

to possess some antimicrobial properties against

pathogenic microorganisms such as Staphylococcusaurens, Bacillus substilis and E.Coli.5,6 However,

the source of the reported antimicrobial activity in

cow dung has not been accounted for. It is likely

that such activity emanates from the proteolytic ac-

tivity of the remnants of gastrointestinal proteases

that assist with the digestion of orally ingested

plant-derived proteins in cows or as metabolic prod-

ucts of microorganisms that feed on the cow dung

material.7 Proteolytic enzymes or proteases such as

collagenases, elastases and matrix-metallopro-

teinase (MMPs) have been implicated in the wound

healing process. Collagenases and elastases are re-

ported to be involved in the debridement (removal)

of wound eschar, a non-viable wound covering that

is the source of infection in wounds. In support of

the notion that collagenase and elastase proteases

are involved in the wound healing process, these en-

zymes are usually added to commercial debriding

ointments that are applied on wounds for the re-

moval of nonviable tissue.6 Matrix-metallopro-

teinase, on the other hand, are involved in the

removal of damaged extracellular matrix (ECM)

that occurs at the edge of acute skin wounds during

the inflammatory stage of the wound healing

process.8 The presence of such proteases in cow

dung could be one of the contributing factors to the

efficacy or presumed effectiveness of the applica-

tion of cow dung in burn wound management. The

current study was therefore aimed at the purification

and characterization of proteases that may be pres-

ent in cow dung in an attempt to validate its usage

in traditional African home burn wound treatment.

Materials and methods

Sample collection and preparationCow dung samples were collected fresh (not dry)

from Onderstepoort Veterinary Institute (Sample 1),

a farm on the Brits Road (Sample 2) and a village

in the North West Province (Sample 3). The sam-

ples were collected using the convenience sampling

method. Two grams of fresh cow dung was sus-

pended in 250 ml of 1M phosphate buffer, pH 7.5,

and then homogenized using a blender. The ho-

mogenate was centrifuged at 5000 RPM for 10 min-

utes. The supernatant was recovered and 200 ml

used in further purification steps.

Screening of cow dung extract for the presenceof protease enzymes

The homogenate supernatant of the three cow

dung samples were screened for the presence of

protease enzymes using a universal protease activity

assay.9 Briefly, 5 ml of 0.65% w/v casein and 5 M

Potassium Phosphate buffer, pH 7.5 solution was

incubated in a water bath at 37˚C for 5 minutes. The

1 ml of homogenate was then added to the casein

solution and incubated for a further 10 minutes at

37˚C. After 10 minutes, 5 ml of 0.11 M TCA solu-

tion was added to the reaction mixture and incu-

bated at 37˚C for 30 minutes. The reaction mixture

was filtered and 5 ml of sodium carbonate and 1 ml

of Folin’s reagent were added to 2 ml of the filtrate.

The solution was then incubated for 30 minutes at

37˚C. The solution was filtered again and ab-

sorbance was read at 660 nm. Then enzyme units

were calculated using the following equation:


105

The cow dung sample that exhibited the highest

specific activity was further purified.

Isolation and purification of protease enzymesAmmonium sulphate fractionation. Proteins in

the cow dung extract were fractionated using the

ammonium sulphate precipitation technique.

Briefly, increasing amounts of ammonium sulphate

were added to the cow dung extract while it was

stirred with a magnetic stirrer and then the mixture

was centrifuged. The pellets obtained 0%, 20%,

40%, 60% and 80% saturation and were re-sus-

pended in saline phosphate buffer and quantified for

protein concentration and protease activity, fol-

lowed by calculation of specific activity and purifi-

cation fold.

Size exclusion chromatography. Pre-swelled

Biogel P100 was packed in a chromatography col-

umn. 2 ml of the samples were loaded onto the col-

umn and eluted using Tris buffer, pH 9. Fractions

were quantified for protein concentration and pro-

tease activity. Also, fractions corresponding to a

particular protein/enzyme chromatographic peak

were combined and assessed for purity, followed by

calculation of specific activity and purification fold.

Anion exchange chromatography. Pre-swelled

Sephadex A250 was packed in a chromatography

column. 2 ml of the samples were loaded onto the

column and eluted using Tris-NaCl buffer, pH 9.

Fractions were quantified for protein concentration

and protease activity. Also, fractions corresponding

to a particular protein/enzyme chromatographic peak

were combined and assessed for purity, followed by

calculation of specific activity and purification fold.

Cation exchange chromatography. Pre-swelled

Sephadex C50 was packed in a chromatography

column. The same procedure as in anion exchange

chromatography was then carried out.

Characterization of purified enzyme fractionsDetermination of molecular weight and purity.

Molecular weight of the ammonium sulphate pre-

cipitation and chromatographic protease fractions

was determined using SDS-PAGE with the Laemmli

gel system.10 In brief, after the preparation and cast-

ing of the gel system (12% separating gel and 4%

stacking gel) aliquots of concentrated protease frac-

tions and molecular weight markers that were pre-

pared and denatured in a sample preparation buffer

containing 2-Mercaptoethanol, glycerol, SDS and

bromophenol blue were loaded into wells created in

the stacking buffer. Electrophoresis was run for 1

hour, then the gels were removed and visualised with

the Coomasie brilliant blue A250 stain. Protein frac-

tions were regarded as pure if they yielded a single

band, and partially pure if they yielded 2 or 3 bands

on SDS-PAGE.

Determination of optimum temperature and op-

timum pH. The optimum pH of the purified or semi-

purified protease enzyme fractions was determined

by incubating the casein-enzyme reaction mixture in

buffers of different pH values, namely; sodium ac-

etate pH 5.5, sodium phosphate pH 6.0, phosphate

buffer pH 7.0, sodium carbonate pH 8.0 and glycine

NaOH pH 9.0. The enzymes were assayed according

to the protease enzyme assay as described earlier.

The optimum temperature of the purified or semi-

purified protease enzyme fraction was determined

by incubating the reaction mixture at 30°C, 35°C,

40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C and

75°C. The enzymes were assayed according to the

protease enzyme assay described above.

Determination of enzyme kinetic constants and sub-

strate specificity. Enzyme kinetic constants, Km and

Vmax, of pure and partially pure protease enzymes

were determined by means of the Lineweaver-Burk

double reciprocal plots. In brief, aliquots of pure or par-

tially pure protease enzymes were incubated with a

range of concentrations (0-2 mg/ml) of collagen, elastin

and haemoglobin for 30 minutes at 37oC. Enzyme re-

action velocities were then determined and their recip-

rocals (1/v) were plotted against the reciprocal of

substrate concentration 1/[S], Vmax was taken as the

reciprocal of the y-intercept and Km was determined

from the slope of the curve. Substrate specificity (i.e.

the ideal or best substrate for a given protease fraction)

where: W = total volume of assay in ml

X = volume (ml) used in the caloric detection

Y = volume (ml) of the enzyme used for the assay

Z = time (minutes) of the assay

Enzyme unit (units/ml) = ------------------------------------------(tyrosine released) (W)

(X) (Y) (Z)


106

was determined by comparing the Km for the hydroly-

sis of collagen, elastin and haemoglobin. For the pur-

pose of the current study, the substrate with the lower

Km was regarded as the best substrate for the protease

enzyme fraction.

Protein quantification. Protein quantity was deter-

mined according to the Lowry assay,11 using the Bovine

Serum Albumin (BSA) as a standard. A series of stan-

dard samples ranging from 0-0.2mg/ml were prepared.

Lowry solution and Folin phenol reagent were added to

each sample and allowed to stand at room temperature

for 25 minutes. Thereafter absorbance was read at 750

nm using the AVARIAN spectrophotometer. The ab-

sorbance readings were used to construct a standard

curve. The test samples were subjected to the same pro-

cedure as the BSA standards and their protein concen-

tration obtained by interpolation.

Enzyme activity assay. Enzyme activity was deter-

mined by the protease enzyme assay protocol as out-

lined earlier, under the screening of the cow dung

homogenate for protease activity.

Results

Screening for the presence of proteases from cowdung

Three fresh cow dung samples collected at different

locations were screened for the presence of crude pro-

tease enzymes using casein as a substrate. All samples

showed some protease activity of no significant differ-

ence to each other. The results of the screening test are

summarized in Table I.

Isolation and purification of protease enzymes fromcow dung

Cow dung Sample 3 was subjected to sequential am-

monium precipitation, size exclusion chromatography

and ion exchange chromatography. The results obtained

after each purification are presented below.

Ammonium sulphate fractionation. The results of the

ammonium sulphate fractionation step are shown in

Table II. The highest protease activity was observed in

the 40% (3.31 units) and 80% (3.40 units) ammonium

sulphate fractions. The samples were labelled fractions

A and B, respectively, and further purified using size

exclusion

Size exclusion chromatography. The 40% and 80%

precipitates (labelled fraction A and B, respectively) ob-

tained during the ammonium sulphate fractionation

step, were dissolved in phosphate buffered saline and

purified using size exclusion chromatography on Biogel

P100, eluted with 0.5 M Tris buffer, pH 9.0. The result-

ant chromatogram is shown in Fig. 1. A single protease

peak corresponding to fraction 2 to 8 was observed on

size exclusion chromatogram of the 40% fraction of

ammonium sulphate. The fractions were pooled to-

gether, labelled fraction C, and further subjected to pu-

rification using anion exchange chromatography. Also,

a single protease peak corresponding to fractions 1 to 6

was obtained from size exclusion chromatography of

the 80% fraction of ammonium sulphate. Fractions cor-

responding to the protease peak were pooled together,

labelled fraction D and further subjected to anion ex-

change chromatography.

Anion exchange chromatography. Fractions C

and D obtained from size exclusion chromatography

of the ammonium sulphate fractions A and B were

loaded onto a Sephadex A250 column, equilibrated

with 0.5 M Tris buffer, pH 9.0. The column was then

eluted with 0.5 M Tris.NaCl buffer, pH 9.0. The re-

sultant chromatogram is shown in Fig. 2. A single

protease peak corresponding to fractions 1 to 3 was

obtained from anion exchange chromatography of

fraction C. These fractions were pooled together,

concentrated with the Amicon centrifugal tubes, la-

belled as fraction E and subjected to further purifi-

cation using cation exchange chromatography. Two

protease peaks corresponding with fractions 1 to 3

and fractions 5 to 6 were obtained from the anion

exchange chromatography of fraction D. Fractions

corresponding to each band were pooled, concen-

trated using Amicon centrifugal tubes, labelled F and

G, respectively, and subjected to further purification

using cation exchange chromatography.

Cation exchange chromatography. Fractions E, F

and G obtained from anion exchange chromatogra-

phy were loaded onto a Sephadex C50 column,

equilibrated with 0.5 M Glycine buffer, pH 10.0, and

the column was then eluted with 0.5 M Glycine-

NaCl buffer, pH 10.0. The resultant chromatogram

is shown in Fig. 3. Single protease peaks correspon-

ding to fractions 7 to 12 from fraction E, fractions 3

to 7 from fraction F and fractions 1 to 9 from frac-


107

Cow dung sample Protein (mg)

Total protease activity (units a)

Specific activity (units/mg)

Sample 1 272.5 ± 0.050 20 ± 0.020 0.07 ± 0.003

Sample 2 287.5 ± 0.002 30 ± 0.003 0.10 ± 0.002

Sample 3 242.5 ± 0.040 30 ± 0.007 0.12 ± 0.001

Table I - Screening for the presence of protease enzymes in cow dung

Ammonium sulphate

concentration

Protein (mg)

Protease activity (units)

Specific activity (units/mg)

0% 272.50 20.00 0.07

20% 106.0 0.02 0.0002

40% (A) 143.9 3.31 0.02

60% 38.75 1.98 0.05

80% (B) 20.7 3.40 0.16

Table II - Ammonium sulphate fraction of protease enzymes from cow dung

Fig. 1 - Size exclusion chromatograms with the corresponding protease activity for each fraction. (i): Fraction A, (ii): Fraction B


108

Fig. 2 - Anion exchange chromatogram with the corresponding protease activity for each fraction. (iii): Fraction C, (iv): Fraction D

Fig. 3 - Cation exchange chromatograms with their corresponding protease activity. (v): Fraction E, (vi): Fraction F, (vii): Fraction G


109

tion G were separately pooled together, concentrated

using Amicon centrifugal tubes and labelled fraction

H, I and J, respectively.

The purity of the protease enzymes was assessed

by the calculation of specific activity, as shown in the

purification tables, Tables III and IV, for fraction A

and B, respectively. Purification was observed to im-

prove with each step until the final purification step.

The specific activity of fraction H was found to be

0.13 units/mg with a purification fold of 1.8 and a

percentage yield of 4.85%. The specific activity of

fraction I after cation exchange chromatography was

0.40 units/mg with 5.71 fold purification and yield

of 9.95%. The specific activity of fraction J after

cation exchange chromatography was 0.68 units/mg

with 9.71 fold purification and yield of 8.15%.

Characterization of the purified protease enzymeThe purified protease enzymes were characterized

according to substrate, their kinetic properties and

substrate specificity, optimum temperature, as well

as optimum pH. The results are presented below.

Kinetic properties and substrate specificity. The

kinetic constants (Km and Vmax) of each of the pu-

rified or partially purified protease enzyme fractions

were determined using the Lineweaver-burk plot for

enzyme activity upon collagen, elastin and haemo-

globin at 37˚C. The results are shown in the plots de-

picted in Fig. 4, Fig. 5 and Fig. 6, respectively.

Fraction H showed higher affinity for elastin (Km:

3.54 mg/ml) and the lowest affinity for haemoglobin

(Km: 75.25 mg/ml). The rate at which fraction H

catalyses the conversion of the substrate to product

was fast with collagen (Vmax: 0.0216 units/ml) and

slowest with elastin (Vmax: 0.0079 units/ml). Frac-

tion I showed higher affinity for haemoglobin (Km:

0.56 mg/ml) and the lowest affinity was observed

with elastin (Km: 9.93 mg/ml). The rate at which

fraction I catalyses the conversion of the substrate

to product was higher for haemoglobin (Vmax:

0.0539 units/ml) and lowest for elastin (Vmax:

0.0040 units/ml). Fraction J also showed higher

affinity for collagen (Km: 1.61 mg/ml) and the low-

est affinity for haemoglobin (Km: 26.49 mg/ml).

The rate of activity for fraction H was higher with

haemoglobin (Vmax: 0.0470 units/ml) and lowest

when reacting with collagen (Vmax: 0.0032

units/ml).

Purification step Total protein

(mg)

Protease

activity

(units)

Specific activity

(units/mg)

Purification

fold

%

yield

Homogenization 272.50 20.00 0.07 1 100

ASF (Fraction A) 143.90 3.31 0.02 0.29 16.6

SEC (Fraction C) 18.03 2.08 0.12 1.7 10

AEC (Fraction E) 10.58 1.48 0.13 1.8 7.4

CEC (Fraction H) 7.54 0.97 0.13 1.8 4.85

Table III - Purification table for protease enzyme isolation from cow dung (Fraction A)

ASF = Ammonium sulphate fractionation, SEC = Size exclusion chromatography, AEC = Anion exchange chromatography, CEC = Cation ex-

change chromatography.


110

Purification

tep

Total protein

(mg)

Protease activity

(units)

Specific activity

(units/mg)

Purification

fold

%

yield

Homogenization 272.50 20.00 0.07 1 100

ASF (Fraction B) 20.70 3.40 0.16 2.29 17

SEC (Fraction D) 10.60 2.72 0.26 3.7 13.6

AEC (Fraction F) 8.02 2.25 0.28 4 11.3

AEC (Fraction G) 5.90 2.13 0.36 5.14 10.7

CEC (Fraction I) 4.93 1.99 0.40 5.71 9.95

CEC (Fraction J) 2.39 1.63 0.68 9.71 8.15

Table IV - Purification table for protease enzyme isolation from cow dung (Fraction B)

Fig. 4 - Double reciprocal plot for fraction H obtained after cation exchange chromatography (Sephadex C50). (A): using collagen as substrate,

(B): using elastin as substrate, (C): using haemoglobin as substrate


111

Optimum temperature and pH. The protease ac-

tivity of fractions H, I and J was investigated at dif-

ferent temperatures and the results are shown in

Fig. 7. The purified enzymes fractions proved to

have different optimum temperatures. The optimum

temperature was found to be 40˚C for fraction H,

35˚C for fraction I, and 36˚C for fraction J. The pro-

tease activity of the purified fractions was investi-

gated at different pH levels in the range of 5 to 9 and

the results are shown in Fig. 8. The three fractions

showed optimum activity at different pH levels. The

optimum pH was recorded to be 6 for fraction H, 7

for fraction I, and 8 for fraction J.

Discussion

The results of the current study indicated the

presence of some proteolytic enzymatic activity in

the homogenate solutions of three cow dung samples

collected at different places in the north of Pretoria,

South Africa. Subsequently, three protease fractions

were purified from the homogenate of one of the

samples using size exclusion-, anion exchange- and

cation exchange- chromatography. The purity of the

fractions was determined using SDS-PAGE which

showed some level of acceptable purity (results not

shown). Furthermore, characterization of the puri-

fied fractions revealed their optimum temperature to

be largely similar (40°C for fraction H and 35°C for

both fractions I and J) and optimum pH to be closer

(pH 6 for fraction H, pH 7 for fraction I and pH 8

for fraction J) for their proteolytic activity. However,

the fractions showed disparities on substrate speci-

ficity for their proteolytic activity. In this regard,

fraction H showed higher affinity for elastin, fraction

Fig. 5 - Double reciprocal plot for fraction I obtained after cation exchange chromatography (Sephadex C50). (A): using collagen as substrate,



112

Fig. 6 - Double reciprocal plot for fraction J obtained after cation exchange chromatography (Sephadex C50). (A): using collagen as substrate,


Fig. 7 - The effects of temperature on the enzyme activity of the purified protease enzyme fractions


113

I for haemoglobin and fraction J for collagen.

The exhibited proteolytic activity and the presence

of the purified protease fractions demonstrated in this

study could contribute to the efficacy or presumed

effectiveness of the usage of cow dung for burn

wound healing or management, which many African

communities rely on. The attempt to establish the

source or origin of proteases in cow dung relied on

the results obtained from the characterization of the

purified fractions. The optimum pH of the purified

fractions tended towards neutrally-alkaline, in the

range of 6 to 8. Most proteases such as pepsin, rennin

and trypsin of animal gastrointestinal origin have an

acidic optimum pH, which rules out the possibility

that the purified protease fractions are of gastroin-

testinal origin. Notably, several proteases with more

similar optimum pH characteristics have been puri-

fied as fermentation products of bacteria or fungi. For

example, a protease with optimum pH of 8 was iso-

lated as a fermentation product of Halomanas spp.12

another protease with optimum pH of 9 was purified

from Pseudomonas spp. in a study by Vijayaragharan

et al. 13 and proteases with optimum pH of 7 were pu-

rified from Bacillus spp. in studies done by

Padmapriya and Williams14 as well as Baechaki et

al.15 As such, it appears that most proteases of micro-

bial metabolic production are of a neutral and alka-

line nature, which resonates with the optimum pH of

the purified fractions observed in the current study.

Conclusion

In conclusion, the current study has demonstrated

the presence of some proteolytic activity in cow

dung that is an important aspect of commercial burn

wound management ointments. The demonstrated

proteolytic activity in cow dung samples could be

due to proteases of microbial origin emanating from

microbes that cow dung plays host to. Such a deduc-

tion could also bring forth a location dynamics phe-

nomenon in the presumed efficacy of the usage of

cow dung in burn wound management that should

be subjected to further investigations. The findings

of the current study may go a long way in mitigating

the tensions around the co-existence of African

home management of burn wounds and the take-

over by modern health practitioners, as a scientific

basis for acceptance of the ancient practice is pro-

vided. This is important as the home treatment for

burn wounds is often the first intervention and also

helps to calm the patient.

Fig. 8 - The effects of pH on the enzyme activity of the purified protease enzyme fractions

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114


Documents

PURIFICATION AND C HARACTERIZATION OF PRO TEASES FROM … · a farm on the Brits Road (Sample 2) and a village in the North West Province (Sample 3). The sam-ples were collected using