EFFL „ î ULTRASONICATION ON POLYPEPTIDE PROFILES OF SKELETAL

MUSCLE PROTEINS OF GaUus gaUus

DISSERTATION

CT p M I j j £ j ^ x . z w . . . .^ Nl^ OF THE REQUIREMENTS FOR THL _ ._ - THE DEGREE OF

ifHasfter of $I)tlos(opI)p in

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UNDER IHE SUPERVISION OF

DR. RIAZ AHMAD

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, External: 2700920/21-3430 Phones { Internal: 3430

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DEPARTMENT DF ZDDLDCBY ALIGARH MUSLIM UNIVERSITY

ALIGARH-202002 INDIA

1. ENTOMOLOGY 2. FISHERY SCIENCE &AQUACULTURE _ ^, , __ 3 GENETICS O. A/o / ZD 4. NEMATOLOGY O' ; 'T^ Q /O/Z 5. PARASITOLOGY Dated..4-.&.A^.y.iY..Q.'.u.

CERTIFICATE

This is to certiiy that the work entitled "Effect of u|traliVm9 t̂)c>tt on polypeptide

profiles of skeletal muscle proteins of Gallus^gallus'^ emboHies the original

research work independently pursued by MJ-; Rashid "'Slaieem^̂ .-u my

supervision. He is permitted to submit the dis;serta^ioi| tofyyat-ds the partial

fulfillment of the requirements of the degree of Master of l̂ 'Hlrosophy in Zoology.

Dn^^iaz Ahmad

Research Supervisor

Assistant Professor Section of Genetics

Department of Zoology Aligarh Muslim University

Aligarh, U.P., India

w

Contents

Page No.

Abbreviation used i

1. Introduction 1̂ 9

2. Statement of the Problem 10

3. Materials and Methods 11-17

3.1 Source of chemicals 11-12

3.2 iVtuscfe source and Its co(/ection 13

3.3 Preparation of Natural Actomyosin (NAM) 13

3.4 Protein estimation 14

3.5 Ultrasonication treatment 14

3.6 Biochemical parameters selected to assess the impact 15

of ultrasonication on Natural Actomyosin (NAM)

3.6.1 ATPase activity determination 15

3.6.1.1 Ca-'ATPase 15

3.6.1.2 Mg^* ATPase 15

3.6.1.3 K^EDTA) ATPase 15

3.6.2 Turbidity determination 16

3.6.3 Solubility determination 16

3.6.4 Sodium dodecyl sulphate polyacrylamide 16-17

gel electrophoresis (SDS-PAGE)

3.6.4.1 Staining of PA-gels 16-17

17

18-24

19-20

19

19

20

20

0

states of different KCl/ NaCI molarities

4.3 Clianges in solubility profiles of NAM in various solubility 21

states of different KCl/ NaCl molarities

4.4 Assessment of submolecular changes in polypeptide 21-23

composition of NAM by SDS-PAGE

4.5 Status of selected parameters during ultrasonication of aged 23-24

NAM

4.5.1 CV-. Mg-- and K*(EDTA) ATPases in aged NAM 23

4.5.2 Changes in solubility profiles of aged NAM 23

4.5.3 Assessment of submolecular changes in polypeptide 24

composition of aged NAM by SDS-PAGE

Figures 25-37

List:

1. Effect of ultrasonication on Ca '̂̂ -ATPase activity of NAM 25

Effect of ultrasonication on Mg^^-ATPase activity of NAM 26

3. Effect of ultrasonication on K*(EDTA)-ATPase activity of NAM 27

4. Variations in turbidity of NAM sonicated under dissolved and 28

Suspended states

5. Effect of ultrasonication on solubility of chicken NAM in different 29

states.

6. Electrophoretic (SDS-PAGE, 10%) profiles of NAM dissolved in 30

0.6 M KCl and its densitogram.

7. SDS-PAGE (10%) profiles ofsonicated NAM diluted to 0.1 M. 31

8. Electrophoretic (SDS-PAGE, 10%) profiles of NAM ultrasonicated 32

in dissolved state and then diluted to 0.2 M NaCl.

9. SDS-PAGE (10%) profiles of NAM diluted to 0.2 M and then 33

sonicated.

10. Effect of ultrasonication on Câ "̂ -, Mg^*- and K^(EDTA) ATPases 34

activities of aged actomyosin.

1 1. Solubility profile of aged actomyosin sonicated following 21 days 35

storage.

12. Electrophoretic (SDS-PAGE, 10%) profiles of supernatant and 36

pellet collected from aged actomyosin.

13. Densilograms of supernatants and precipitates of SDS-PAGE proHles 37

of Fig. 12.

6. Discussion 38-42

7. Summary 43-44

8. Bibliography 45-53

9. Acknowledgements ii-iii

10. Appendix iv-v

Abbreviations used

ANSA

ATP

BPB

BSA

CBB

EDTA

kD

KHz

MyHC

NAM

PAGE

PMSF

SDS

TEMED

1 -Amino-2-naphthol-4-sulphonic acid

Adenosine 5' triphosphate

Bromophenol blue

Bovine serum albumin

Coomassie brilliant blue

Ethylene diamine tetraacetic acid

Kilo Dalton

Kilo hertz

Myosin heavy chain

Natural Actomyosin

Polyacrylamide gel electrophoresis

Phenylmethane sulphonylflouride

Sodium dodecyl sulphate

N,N,N' N'-Tetra methyl ethylene diamine

Cfiapter 1

Introduction

There are 3 main constituents of vertebrate muscle: sarcoplasmic proteins,

myofibrillar proteins and collagen (Lawrie, 1998). Sarcoplasmic protein repertoire is

made up by most of metabolic enzymes, proteases and some other water soluble

proteins (Babji and Kee, 1994). In addition to their metabolic function, sarcoplasmic,

proteins are of organoleptic value concerned with food taste. Collagen contents impart

textural toughness on cooking and are of concern in cattle meat rather than chicken or

fish. Due to their relative proportion, function and edible value, myofibrillar proteins

are most important. Myofibrillar proteins constitute about 50 to 60% of the total

skeletal muscle proteins, most of which are high ionic strength soluble and a small

proportion of them are water soluble proteins (Goll et al., 1977). Apart from being

responsible for contraction and relaxation, myofibrillar proteins account for muscle

toughness or rigor and ~97% of muscle water holding capacity, an important quality

during post-mortem storage.

An important factor in consumer acceptability of meat is degree of

tenrlerness whirh k ln«:t tn varvintr Hearees during post-mortcm aging and Storage.

f skeletal muscle, myofibrillar proteins and

rmine the

IS calpain

egradatioi

5; Kemp

to produ(

)n (Chrys

1992) an

:al force 1

as and G(

Studies show increase in tenderness with low frequency ultrasound (20-24 KHz)

treatments (Dolatowski, 1989; Dickens et al., 1991). The tenderness has been

attributed to 2 factors : (1), Release and activation of tenderness causing endogenous

proteases (calpains and cathapsins etc.) from lysosomes (Koohmaraie, 1988; 1996);

(2), fragmentation or solubilization of myofibrillar proteins and other cellular

components (Dolatowski, 1988; Ito et al., 2003; 2004). Few reports have dealt with

isolated individual proteins of myofibrillar system and available ones are fairly old.

For instance, enzymatic activity of myosin decreases with ultrasonic treatments

(Barany et al., 1963) whereas actin has been stable under ultrasonication treatment

(Asakura, 1961).

Myosin, actin and a number of other proteins are well characterized

constituents of Natural Actomyosin (NAM), the major contractile complex of

myofibrils soluble at high ionic strength salt solutions. SDS-PAGE profiles reveal the

occurrence of only trace components other than actin and myosin and the regulatory

proteins, troponins and tropomyosin (Porzio and Pearson, 1977; Huang et al., 2009;

Vasilleva et al., 2010). The perceived changes can be monitored by several

established parameters of well characterized proteins and polypeptides in the

extracted skeletal muscle NAM or myofibrils.

(a) Myosin molecule Flexible hinge region

Myosin head

(b) Portion of a thick filament

Thick filament

.

The contractile apparatus (essentially NAM) of myofibrils consists of

approximately eight major proteins which include myosin, actin, tropomyosin,

troponins, C-proteins, M-proteins, a-actinin and p-actinin. For a better understanding,

a brief description of the constituent with reference to chicken {Gallus gallus)

myofibrils or NAM is given below:

Myosin

Myosin is the major and functionally central protein of myofibrils. It is a

hexamer with two heavy chains and four light chains. N-terminals of twin heavy

chains form two globular heads. The ATPase activity and actin binding sites are

located to two heads (Levitsky, 2004). Upon tryptic digestion myosin molecule is

cleaved into two major subfragments, light meromyosin (LMM) and heavy

meromyosin (HMM). Heavy meromyosin retains the ATPase activity and actin

binding properties of myosin where as light meromyosin retains the solubility

characteristics of myosin (Harrison et al., 1971; Margossian and Lowey, 1982).

Myosin is the principal contractile proteins of vertebrates and constitutes

bulk of skeleletal muscle. Myosin at low ionic concentration (

and stack as low molecular polypeptides. They have been customarily categorized

into alkali-1 light chain, alkali-2 light chain and DTNB light chain which on SDS-

PAGE shows -21 kD, -16.5 kD and -19 kD subunits, respectively.

Actin

Actin constitutes about 15-20% by weight of total myofibrils. Actin can exist

in two forms, the globular G-actin and fibrous F-actin. G-actin consists of relatively

small globular units while F-actin is a double chain formed as a result of end to end

aggregation of the globular units (Lawrie, 1974). In vivo actin exists as F-actin

(Huxley, 1960). Actin is the major protein of thin filament each containing 340 to 380

molecules of actin. Actin molecules are assembled into asymmetrical filaments in a

front to back manner. This orientation has been found essential for muscle

contraction. The most important properties of actin appear to be G- to F- actin

transformation and interaction with myosin to form actomyosin (Poglazov, 1966). The

transformation of G-actin to F-actin occurs when ionic strength is raised from 0.01 to

0.15 M and is enhanced by Mĝ ^ and Câ * (GoU et al., 1977). On SDS-PAGE, actin

migrates down the gel as a 42 kD component. The actin molecule contains only one

polypeptide chain and is much simpler than myosin.

Actin and myosin aggregates together to form molecular motor, the

actomyosin, which is responsible for muscle contraction (Huxley, 1960). Actomyosin

is the predominant protein of post-rigor meat where as myosin is the major protein of

pre-rigor meat. Natural Actomyosin (NAM) is a complex of actin, myosin, troponin,

tropomyosin and alpha-actinin (Asghar et ai., 1985). The binding of myosin to actin is

reversible which depend on temperature and ionic strength (Laki et al., 1952). In

addition to troponin and tropomyosin, calcium ions are essential for the effective

interaction of actin and myosin during the muscular contraction (Gyorgyi, 1975). The

cycle of contraction and relaxation is coupled with the formation and breakage of

cross-bridges, binding of ATP hydrolysis product (ADP+P/) along with Mg "̂ to

myosin head, dissociation of the complex and replacement by ATP. Initiation of

contraction and fine tuning are regulated by Câ"*̂ levels. Excellent literature is

available on several aspects of actomyosin and myosin ATPase (Tonomura and

Takeshita, 1972; Tonomura, 1986), structure of myosin (Harrington and Rogers,

1984) and its interaction with actin filaments, regulatory proteins (Ebashi et al., 1980)

and, electron microscopic structure of myofibrils (Huxley and Faruqui, 1983).

M-proteins

M-protein was relatively late discovery to the group of myofibrilar proteins.

Although it was earlier referred to as 'M-substance' by Masaki et al. (1968). M-

protein is one of the minor components of thick filaments. M-protein antibodies

strongly stained the middle of A-band (Trinick and Lowey, 1977), thus giving

credence to the belief that M-protein originates from M-line which runs through the

middle of A-band. SDS-PAGE profiles show M-protein stacking between myosin and

C-protein as a 160-170 kD band, respectively (GoU et al., 1977; Trinick and Lowey,

1977).

C-proteins

C-protein is one of the minor components of myofibrillar proteins

constituting about 2 to 3% by weight of myofibrils (Offer et al., 1973). The protein

consists of a single polypeptide chain of molecular weight of 140 kD. C-protein is

presumed to be the component of the thick filaments as it strongly binds to myosin at

low ionic strength. C-protein has no ATPase activity and does not affect the ATPase

activity of pure myosin (Offer et al., 1973).

a-actinin

a-actinin was originally discovered by Ebashi and Ebashi (1965) and

constitutes about 2 to 3% by weight of myofibril, a-actinin is located in Z-band and

functionally it is involved in cross-linking of actin (Briskey and Fukazawa, 1971). It

has been shown to be a component of nearly 100 kD (Suzuki et al., 1976).

P-actinin

It is one of the minor proteins of thin filaments and is believed to be involved

in development of muscles rather in muscle contraction (Ebashi and Kodama, 1966).

Electrophoretically, P-actinin migrates as an 800 kD component on SDS-PAGE (Hay

etal., 1973).

Tropomyosin

Tropomyosin was initially discovered by Bailey (1946; 1948a; 1948b) and

when prepared according to his procedure, the extract is referred to as Bailey

tropomyosin or tropomyosin B to differentiate it from tropomyosin A. Tropomyosin

has no ATPase activity and has been shown to decrease in viscosity with increasing

salt concentration (Bailey, 1948b). Functionally, tropomyosin along with troponin has

been found to possess regulatory influence on calcium ions during muscle contraction

and relaxation (Murray and Weber, 1974).

Troponin

Troponin has been found to possess the molecular weight of around 72 kD

and constitute approximately 4 to 6% of myofibril by weight (Goll et al., 1977).

Troponin is a complex of three different sub-units (Troponin-T, Troponin-! and

Troponin-C) with different physiological roles (Greaser and Gergley, 1971),

Electrophoretically the three subunits of troponin migrate on SDS-PAGE as described

by Goll et al. (1977) and Hay et al. (1973) as follows: 30.5 kD (Trponin-T), 20.8 kD

(Troponin-I) and 17.8 kD (Troponin C) subunits.

The above mentioned constituents are either involved to undergo changes

during external or endogenous factors affecting tenderness of muscle or meat

(Koohmarie, 1996). Some of the common changes which have been recorded in case

of myofibrils lead to loosening of Z-disc and ultimately to disintegration of

sarcomeres (Goll et al., 1977; Takahashi, 1992). In case of both myofibrils and

actomyosin, generally it is degradation of myosin heavy chain or some other

constituent polypeptide. Ultrasonication, in particular, is known to produce a novel

fragment of 30 kD, apart from some other changes (Lyng et al., 1997).

The contribution of ultrasonic radiation alone in tenderizing whole muscle

slices or myofibrils cannot be demarcated while working with intact myofibrils.

Investigations on isolated proteins or extracted NAM complex may help to better

understand mechanism of the radiation. Such studies will exclude the action of

endogenous proteases released during ultrasonic treatment of muscle or myofibrils/7er

se. There are initial indications that NAM may be a suitable model to study

mechanisms and interactions of changes induced by ultrasonication (Ahmad and

Hasnain, 2013a), since in vitro NAM retains several attributes of contractile apparatus

(encased in myofibrils), like ATP splitting, Ca^^-sensitivity, actin binding and

filament formability at low ionic strength, which are typical and essential requirement

of muscle contraction in vivo.

The present study was carried out on natural actomyosin (NAM) of broiler

chicken (Gallus gallus) with the objective of understanding the events initiated or

propagated during ultrasonic exposure. Attempts are made to gain insight by

comparing the effect of ultrasonication on biochemical characteristics such as ATPase

activity, solubility, turbidity and fragmentation pattern by SDS-PAGE profiling.

Cfiapter 2

Statement of the (ProStem

10

It is evident from tlie above literature-review that ultrasonication can be

applied on muscle to increase the commercial acceptance of meat products or in

myofibrillar state constituent proteins can be solubilized to produce 'formed meat'

products. However, individual contribution of endogenous proteases and the

ultrasonication, the two main tenderizers of meat, is yet to be demarcated. To avoid

interference of endogenous protease activities, instead of intact myofibrils natural

actomyosin (NAM) of broiler chicken {Gallus gallus) was preferred as a model

system. There were two main objectives:

1. To demonstrate if low intensity ultrasonic radiation (20 KHz) dissociates,

fragments or solubilizes Natural Actomyosin (NAM) of chicken, Gallus

gallus.

2. While undergoing the above mentioned probable change(s), NAM has to be

in an insoluble (as it exists in vivo) or in a soluble state at high salt

concentration.

It was envisaged that the change(s) in NAM interaction will be reflected in

Câ "̂ , Mĝ "̂ and K^(EDTA) activated ATPases, the dissociation or structural integrity

will be reflected in loss of opacity (turbidity) and solubility, and changes in

submolecular polypeptide composition in SDS-PAGE profiles.

CfiapterJ

MateriaCs aiuf Metfiods

11

3.1 Source of Chemicals

Name of the chemical

1 -Amino-2-naphthol-4-sulphonic acid

Acrylamide

(USA)

Adenosine 5'-Triphospliate

(USA)

Ammonium molybdate

Ammonium persulphate

(USA)

Bis-acrylamide

(USA)

Bovine serum albumin

Bromophenol blue

Calcium chloride

Coomassie brilliant blue R250

Copper (II) sulphate pentahydrate

Ethylenediamine tetraacetic acid (EDTA)

Ethanol

Folin & Ciocateu's phenol reagent

Glacial acetic acid

(India)

Glycerol

Hydrochloric acid

Magnesium chloride

Maleic acid

Methanol

Perchloric acid

Phenyl methane sulphonyl fluoride

Potassium chloride

(USA)

Sodium bisulphate

Source

CDH (India)

Sigma-Aldrich

Sigma-Aldrich

Qualigens (India)

Sigma-Aldrich

Sigma-Aldrich

SRL (India)

SRL (India)

SRL (India)

Merck (India)

SRL (India)

Merck (India)

SRL (India)

Merck (India)

Loba Chemie

Fisher Scientific

Fisher Scientific

Qualigens (India)

SRL (India)

Merck (India)

Merck (India)

Merck (India)

Sigma-Aldrich

Fisher Scientific

12

Sodium carbonate

Sodium chloride

Sodium hydroxide

Sodium dodecyi sulphate

Sodium potassium tartarate

Sodium sulphite

Sulphuric Acid

TEMED

(USA)

Trichloroacetic Acid

Tricine

(USA)

Tris (Hydroxymethyl) Aminomethane

Fisher Scientific

CDH (India)

Qualigens (India)

SRL (India)

Fisher Scientific

CDH (India)

Merck (India)

Sigma Aldrich

Fisher Scientific

Sigma-Aldrich

SRL (India)

Other chemicals of routine use such as acids, bases and salts etc. were of AR

Grade.

13

3.2 Muscle source and its collection

Local venders were approached to purchase the breast muscle of chicken

(Gallus gallus). The muscle was transported under ice from the nearby market. Breast

muscle was excised out carefully avoiding any other contamination using sterilized

knife and scissors. The muscle was weighed every time and processed for actomyosin

preparation.

3.3 Preparation of natural actomyosin (NAM)

Natural actomyosin from chicken breast muscles was prepared by the

modified protocol described by Ahmad and Hasnain (2006). Fresh muscles from the

breast of broiler (3 months of age) were excised out. Muscles were chopped, minced

with sharp knife and thoroughly washed. Minced muscles were washed at least three

times with phosphate buffer (50mM, pH 7.1). These washed muscles were kept

overnight in extraction buffer (0.6M KCl in 20mM Tris-maleate buffer, pH 7.1). The

viscous extract was squeezed manually through muslin cloth to separate out insoluble

connective tissue. Squeezed viscous extract was centrifuged at 5000 rpm, 15 minutes

at 4''C. After centrifugation NAM was precipitated out by diluting the extract with

chilled distilled water and centrifuged at 5000 rpm, 20 minutes at 4°C. Precipitates

thus obtained were dissolved in dissolution buffer (0.6M KCl in 20mM Tris-maleate,

pH 7.1). In order to remove traces of free myosin, 2 to 3 washes were given in 0.2M

KCl (20mM Tris-maleate pH 7.1). NAM dissolved in solvent (0.6M KCl; Tris-

maleate, pH 7.1) were precipitated at 0.06M KCl (20mM Tris-maleate, pH 7.1) by

distilled water. This step was repeated two times to remove low ionic salt soluble

impurities. All solutions and distilled water used in preparation were chilled and at

each extraction step PMSF were added to minimize the effect of proteolysis enzymes.

14

Finally NAM was dissolved in solvent buffer, dialyzed overnight against the same

buffer, cleared by centrifugation at 10,000 rpm and stored in crushed ice for further

investigations.

3.4 Protein estimation

Protein concentrations of NAM for various analyses were determined by the

Biuret method of Gornall et al. (1948) taking bovine serum albumin as standard.

Absorbance (optical density) was recorded at 520nm on BioSyn UV-Visible

spectrophotometer. All the values were the means of five estimates.

3.5 Ultrasonication treatment

Fixed amounts (10ml) with known concentration of NAM dissolved in 0.6M

KCl and suspended in 0.2M, 0.3M and 0.5M KCl solutions were treated with low

intensity ultrasonic radiation (20 KHz). Samples of actomyosin for ultrasonic

treatment were kept in ice beaker. Ultrasonic treatment was given by Ralco

immersible probe ultrasonicator. Actomyosin was ultrasonicated for a total of 30 min

and four aliquots of equal volume were collected at the time intervals of 5, 10, 20 and

30 min. the probe of sonifier was immersed in the middle of the beaker and

ultrasonication bursts of 15 seconds followed with 10 seconds interval of additional

cooling lag.

15

3.6 Biochemical parameters selected to assess the impact of ultrasonication

on NAM

3.6.1 ATPase activity [Ca^^, Mg^^ andlC(EDTA)-ATPase]

Assays for all ATPases were carried out at 20°C and liberated inorganic

phosphate was calculated by the method of Fiske and Subbarow (1925). Absorbance

was recorded at 640nm on Biosync UV-Visible spectrophotometer.

5.6.1.1 Ca^^ - ATP ase activity

It was assayed in a reaction mixture of 2ml at a final concentration of 5mM

CaCb, ImM ATP, 25mM Tris-HCl buffer (pH 7.5) and NAM with protein

concentration of 4mg/ml. The reaction was stopped and protein was precipitated with

addition of 15% Trichloroacetic acid. Subsequently, colour of released ?/ by myosin

after the addition of l-Amino-2-naphthol-4-sulphonic acid (ANSA) was read after 30

min incubation at 37°C.

3.6.1.2 Mg^^-A TPase activity

It was assayed as described above except MgCb was substituted for CaCb of

the same molarity. Other components were of same molarity and pH as used in Câ ^

ATPase activity determination.

3.6.1.3 It (EDTA)-A TPase activity

It was estimated in presence of chelating agent, EDTA in final concentration

of 5mM. MgCb and CaCb were not added in this assay. Other difference was that

final KCl concentration in the assay mixture was 0.06M.

16

3.6.2 Turbidity measurement

Turbidity of the actomyosin during course of ultrasonication was measured

spectrophotometrically by noting changes in absorbance at 320nm on BioSyn UV-Vis

spectrophotometer.

3.6.3 Solubility determination

The samples of actomyosin after ultrasonication and dilutions were

centrifuged at 10,000 rpm for 15 min. Protein concentration in the supernatant was

determined according to Biuret method described above using bovine serum albumin

as standard. The solubility was calculated as the ratio of protein concentration in the

supernatant after centrifugation and total protein concentration of actomyosin before

centrifugation (Liu et al., 2011).

Solubility % = Cs/CoX 100

Where, Cj is the protein concentration in the supernatant after centrifugation

and Co is the protein concentration before centrifugation.

3.6.4 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Ultrasonicated samples of NAM were monitored by SDS-PAGE to observe

dissociation/fragmentation essentially in discontinuous buffer system of Laemmli

(1970). The supernatants and precipitates of sonicated actomyosin, were processed for

electrophoretic profiling. The process was done in denaturing condition where the

polyacrylamide gels, running buffers and samples had SDS in 0.001%, 0.001% and

2% respectively. Equal amounts of proteins were loaded in the gels. The gels were run

initially at 5mA till tracking dye entered into separating gel. The current was then

maintained at 15mA throughout the entire duration of electrophoresis.

3.6.4.1 Staining ofPA-gels

17

Following overnight washing of SDS in 5% acetic acid, proteins were stained

with Coomassie Brilliant Blue R-250 dissolved in methanol: acetic acid: distilled

water (5:1: 5). Destaining was carried out in 5% acetic acid. Silver staining was also

performed according to Nesterenko et al. (1994) for selected gels to visualize the light

chains.

3.7 Documentation, densitometry and quantitative assessment of the

obtained data

Stained PA-gels were documented using SONY CYBERSHOT digital

camera (Zoom-4X, 14.1 Megapixels) and by direct scanning on an all-in-one HP

Deskjet (F370) computer setup. Data from both the records were used for

densitometric analysis through software. Densitometry of the selected gel-scans was

done using Scion Imaging (Scion Corporation; Beta release 4.0). Molecular weight

estimates of the gels were carried out on Gel Pro software (Media Cybernetics, USA).

Standard error and other statistical values for biochemical parameters calculated as

per standard formulae.

Cfuipter4

18

During the present study, Natural Actomyosin (NAM) from pectoralis major

muscle of chicken (Gallus gallus) breast was given ultrasonic treatment at 20 KHz in

three states; (i), when NAM was completely soluble (0.6M KCl in 20mM Tris-

maleate; pH, 7.1); (ii), sonicated soluble NAM was diluted to 0.2, 0.3 and 0.5M; and

(iii), NAM was diluted to 0.2, 0.3 and 0.5M KCI/NaCl prior to ultrasonic treatment.

NAM below 0.6 M of these salts exists in insoluble or partially soluble states.

Moreover, the effect of ultrasonication was also evaluated on aged NAM of G. gallus

following storage of 21 days to establish the difference in the effect of sonication on

freshly prepared and aged NAM.

In this write-up, unless otherwise mentioned, NAM refers to chicken breast

muscle natural actomyosin and breast muscle NAM to pectoralis major muscle of

chicken {Gallus gallus) breast. Effect of sonication on the NAM status was analyzed

using the following biochemical/ molecular parameters:

(1) Changes in ATPase activities: (a) Ca^"'-ATPase, (b) Mg^ '̂-ATPase and (c)

K^(EDTA)-ATPases.

(2) Changes in turbidity profiles of NAM in various solubility states at different

KCl/NaCl molarities.

(3) Changes in solubility profiles of NAM in various solubility states at different

KCl/NaCl molarities.

(4) Assessment of submolecular changes in polypeptide of NAM by sodium

dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

(5) Status of selected parameters during ultrasonication of aged NAM

preparations.

19

The results obtained as per these outline and schedule are presented below :

4.1 Comparison of ATPase activities of ultrasonicated NAM under selected

conditions

4.1.1 Ca^^-A TPase activity

Fig. 1A shows the changes in Ca^ -̂ATPase profiles, when chicken NAM was

ultrasonicated in soluble state (0.6 M KCl) and diluted post-sonication. A comparison

with Fig. IB (dilution prior to sonication) reveals that in either of the figures at 10

min of ultrasonic treatment Ca^ -̂ATPase show highest value, if KCl concentration

was 0.5-0.6M. Ultrasonic treatment beyond 10 min brings about a decrease in the

ATPase activity. As for the trend, there exists a general similarity, no matter NAM

was diluted prior to or after the sonication. Similarly, the sonication-duration-

dependent Ca^^-ATPase profiles of NAM at 0.2-0.3M KCl followed a shared trend of

decrease in enzymatic activity. In general, a decrease of the order of 20-25% was

recorded in Câ "̂ -ATPase activities at different molarities (Fig. 1).

4.1.2 Mg^^-A TPase activity

Similar to Ca^^-ATPase, Mĝ "" activated ATPase showed a decline after 10

min of ultrasonication, no matter dilution to 0.2-0.5 M KCl was made prior to

ultrasonication (Fig. 2A) or after this treatment (Fig. 2B). The magnitude was,

however, relatively higher (30-35%). Behavior of NAM at 0.5-0.6M KCl, where it is

either partially or fully soluble, was somewhat different. Slight reduction or activation

of Mg^ -̂ATPase was also characteristic of ultrasonic treatment at 10 min. This

suggested that response to ultrasonication has a correlation with the state of NAM

solubility.

20

4.1.3 K+(EDTA)-A TPase activity

Relatively less decrease occurred in K^(EDTA)-ATPase activity of chicken

NAM when sonicated for 10 min in dissolved state at 0.6M KCl. However, larger

reduction in relative ATPase activities occurred at dilutions of 0.2 and 0.3M KCl. In

addition, dilutions at these two concentrations increase of activity to the extent of

-45% followed in samples treated for 10 min onward (Fig. 3B). The response of

K.̂ (EDTA)-ATPase of sonicated NAM in either cases (Fig. 3A and B) was quite

resembling at 0.5 and 0.6M KCl.

4.2 Changes in turbidity profiles of NAM in various solubility states at

different KCl/NaCl molarities

NAM is fairly turbid or opaque protein complex and any decrease (clearing

effect) in this parameter indicates dissociation of the complex or fragmentation of

constituent polypeptides. On the other hand, an increase in turbidity is an indication of

denaturation or aggregation. By turbidity measurements of sonicated NAM samples,

the underlying idea was to detect which of the above mentioned changes

(dissociation/fragmentation or denaturation) occur during ultrasonication for different

timings. The changes in turbidity (absorbance at 320nm) profiles of NAM diluted

prior to ultrasonic treatment and the NAM samples diluted post-sonication are shown

in Fig. 4. In general with increasing time of sonication, the trend of a constant decline

in the turbidity of NAMs was observed. Compared to control (1.9 OD units at

320nm), turbidity of NAMs declined by -40% during the initial 10 min of sonic

treatment. It showed further decrease of-25% at the end of 30 min of sonic treatment.

The change in the turbidity profile of NAM was independent of the state of protein's

solubility i.e. whether NAMs was diluted prior to sonication or after the treatment.

21

4.3 Changes in solubility profiles of NAM in various solubility states at

different KCl/NaCl molarities

As shown in Fig. 5, tlie effect of ultrasonication on solubility profiles of

chicken NAM was also determined at different KCl concentrations. A solubility

increase of about 50% was apparent at ionic strengths of 0.2 and 0.3M within 10 min

of ultrasonication. Thereafter, the increase remains insignificant up to 30 min of

sonication. The above findings were independent of the state of NAM whether it was

diluted before ultrasonic treatment or diluted after such treatment (Fig. 5A and B). It

is also clear that the NAM in either state remains insoluble at O.IM salt concentration

and not affected by ultrasonication. SDS-PAGE profiles of supernatants collected

from sonicated NAMs of O.IM samples and concentrations above it are compared in

Fig. 7-9.

4.4 Assessment of submolecular changes in polypeptide composition of

NAM by SDS-PAGE

Ultrasonicated NAM samples were analyzed using 10% gels in

discontinuous buffer system of Laemmli (1970), with some modifications described

under Materials and Methods. Firstly, ultrasonicated NAM dissolved in routine

solvent (0.6M NaCl+20 mM Tris-maleate buffer, pH 7.1) was analyzed by SDS-

PAGE. As shown in Fig. 6 (top figure) and its densitogram (below), there are no

definite changes in the submolecular polypeptide composition or their relative

intensities. The major polypeptides in NAM obtained from pectoralis major of Gallus

gallus demonstrated the presence of Myosin Heavy Chain (MyHC~200 kD), a-actinin

(-110 kD), actin (-46 kD) and tropomyosin (39 kD) as the major activity bands.

Proteins having molecular weight smaller than tropomyosin were troponin-I (-23 kD),

22

troponin-C (-18 kD) and at least three myosin light chains (-25 kD, -20 kD and -16

kD). Two LCs are known to costack.

Focus was then shifted to polypeptide analysis of various fractions. It was

apparent from Fig. 5 that solubility of NAM constituents was minimal at O.IM NaCl/

KCl, while no significant differences were there from 0.2-0.3M. Fig. 7 demonstrates

the SDS-PAGE profiles of O.IM KCl soluble polypeptides. It is obvious that the

number of components in supernatant from NAM diluted after sonication were

qualitatively and quantitatively higher (Fig. 7A) than those from NAM diluted prior to

ultrasonication (Fig. 7B). In either case insoluble material (pellet) was removed by

centrifugation. However, even after 30 min of ultrasonication, no marked change in

polypeptide composition of O.IM supernatant is shown by totally soluble NAM {i.e.

0.6M KCl).

Fig. 8 shows SDS-PAGE profiles of NAM samples partitioned into

supernatant and pellet after ultrasonication in 0.6M KCl (dissolved state) and

subsequently diluted to 0.2M. The lanes in both photographs (A and B) clearly

demonstrate the effect of ultrasonication in shifting the solubility of myosin heavy

chain (MyHC=200kD) with the increasing duration of the treatment from high salt

soluble state to low ionic strength solubility. This is evident from the gradient-like

increase in the intensity of polypeptide bands from lane 3-6 in Fig. 8A. A

proportionate decrease in each corresponding lanes of Fig. 83 is also obvious. In

addition to MyHC, changes which fit this description can be seen in bands of actin

and tropomyosin. These polypeptides declined in the pellets of the corresponding

samples with increase in time of sonic exposure.

Although the increase in low-ionic strength protein-component with the

increasing duration was not as prominent as with the parthioned samples of NAM

23

sonicated in 0.6 M (Fig. 9), the process of ultrasonication-induced-solubiiity shift of

actomyosin proteins was apparent even when the NAM was diluted first to 0.2M and

ultrasonicated afterwards (Fig. 9). In comparison with the pellets (Fig. 9B), this is

being better displayed by the supernatant (Fig. 9A). The supernatant was obtained

following centrifugation of sonicates by diluting NAM (suspended or partially

dissolved in 0.2M) with the buffer of the same molarity and ionic strength; i.e., 0.2M

containing 20 mM Tris-maleate of pH 7.1.

4,5 Status of selected parameters during ultrasonication of aged NAM

4JJ Ca^\ Mg^^- and Jt-(EDTA)-ATPases in aged NAM preparations

In contrast to the results obtained with fresh preparations, response of an

aged NAM preparation to ultrasonication at 20 KHz was different (Fig. 10). As shown

in the figure, sonication indiscriminately caused an overall decline of almost 50% of

Câ ""-, Mĝ ""- and K^(EDTA)-ATPase activities within 30 min (Fig. 10). The gradients

of the decline of all three ATPases were also parallel.

4.5.2 Changes in solubility profiles of aged NAM

Compared to fresh preparations, aged NAM shows different changes in

solubility when diluted to O.IM after sonication (Fig. 11). Fresh NAM is almost

insoluble at O.IM salt concentration and sonication does not affect its solubility in low

ionic solutions (Fig. 5). But sonication of aged NAM for 30 min cause -90% increase

of solubility in O.IM salt solution. Rapid increase (-45%) of solubility occurs

between 10 to 20 min of sonication. Beyond 20 min of sonication, solubility increase

is insignificant.

24

4.5.3 Submolecular changes in polypeptide composition of aged NAM by SDS-

PAGE

A comparative of solubility graphs depicted mariced difference in solubility

of fresh and aged NAM in Tris-maleate buffered O.IM KCl (Fig. 5 & 11). SDS-PAGE

of aged NAM (Fig. 12) demonstrates the fraction of polypeptides soluble at O.IM salt.

In comparison to fresh NAM, the supernatant of aged NAM contains more number of

polypeptides. SDS-PAGE of supernatant from aged NAM clearly shows the effect of

sonication (Fig. 12). Lanes 2-5 show two polypeptides of molecular weights ~l42kD,

and ~129kD corresponding to heavy meromyosin (HMM) and rod, as indicated in

other reports (Hasnain and Yasui, 1986; Hasnain and Ahmad, 2006; Ahmad and

Hasnain, 2013b), in sonicated samples at all the timings (5-30 min) of the treatment.

Polypeptides of molecular weights ~95kD (S-1 heavy Fragment) and ~84kD (Light

meromyosin) were observed in the NAM sample sonicated for 30 min (Lane 5). No

such bands were observed in fresh NAM diluted to O.IM salt concentration after or

before sonication. Only proteases are known to release fragments of corresponding

molecular weights.

25

c E

2 Q.

H— O

E •D

(0 a> jq

d. o

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

-•-0.2M -0.3M -A-0.5M •0.6M

Diluted to indicated KCi concentrations after sonication

10 20 30 Duration of uitrasonication (min)

0.3

0.35 c E "c

e 0.25 Q.

° 02 E I 0.15

I °'' i^ 0.05 O

i 0

•0.2M * 0.3M 0.5M

B

0.6M

Diluted to indicated KCI concentrations prior to sonication

10 20 30

Duration of ultrasonication(min)

Effect of uitrasonication on Ca'^-ATPase activity of chicken Natural Actomyosin (NAM). NAM

was dissolved in KCI (with 20mM Tris-maleate; pH, 7.1).

(A) Changes in ATPase activity of sonicated NAM after diluting to 0.2, 0.3 and 0.5M KCI

with Tris-maleate buffer (20mM; pH, 7.1).

(B) Changes in ATPase activity when NAM was sonicated after dilution to 0.2, 0.3 and 0.5M

KCI (in Tris-maleate; pH, 7.1).

NAM dissolved in 0.6 M KCI (20mM Tris-maleate; pH, 7.1) was used as control in either case.

Final molarities of ATPase assays included molarity of solvent KCI buffer which dissolved or

suspended NAM.

2ml reaction ATPase assay mixture contained 5mM CaCl2, 1 mM ATP, 25 mM Tris-HCl buffer

(pH 7.5) and 0.03M KCI at final concentration. Assay temperature was 25°C.

26

^ 0.25

I i 0.2 o a. CO

E •o

^ 01 0)

ja

K 0.05 o

=̂ 0

•^-0.2M •0.6M

Diluted to indicated KCI concentration after sonication

10 20 30

Duration of ultrasonication (min)

0.25 c

I i 0.2 2 a o 0.15 •D

B 0.1 to

^ 0.05

B

•0.2M 0.3M -0.5M 0.6M

Diluted to indicated KCI concentration prior to sonication

10 20 30 Duration of ultrasonication(min)

Effect of ultrasonication on Mg^'-ATPase activity of chicken NAM.

(A) Changes in ATPase activity of sonicated NAM after diluting with 0.2, 0.3 and 0.5M by

Tris-maleate buffer (20mM; pH, 7.1).

(B) Changes in ATPase activity of NAM when NAM was sonicated after dilution to 0.2, 0.3

and 0.5M KCI (in 20mM Tris-maleate; pH, 7.1).

Other experimental parameters were the same as in Fig. I except that Mg"'* was used as the

activator ion instead of Ca'*.

27

0.25

I 0,2

o E =5

0.15

0.1

t^ 0.05 O

c b

(I)

o Q. "B O)

F T3 0) ffl (1> ^ ix •̂— o S

0.3

0.25

0.2

0.15

n 1

0.05

0

-•-0.2M m 0.3M 0.5M -0.6M

B

Diluted to indicated KCI concentration after sonication

10 20 30 Duration of ultrasonication(min)

0.5M -0.6M

Diluted to indicated KCI concentration prior to sonication

10 20 30

Duration of ullrasonication(min)

Fig. 3: Effect of ultrasonication on K^-(EDTA)-ATPase activity of chicken NAM.

(A) Changes in ATPase activity of sonicated NAM after diluting to 0.2, 0.3 and 0.5M by Tris-

maleate buffer (20mM, pH 7.1).

(B) Changes in ATPase activity of NAM when NAM was sonicated after dilution to 0.2, 0.3

and 0.5M KCI (in 20mM Tris-maleate; pH, 7.1).

Other parameters were same as in Fig. 1 except that EDTA replaced Mg'^ or Câ *.

28

E c o eg CO

3

1,6

1.4

1.2

1

0.8

0.6

0.4

0.2

• 0.2M • 0.3M A0.5M • 0.6M

Diluted to indicated KCI concentration after sonication

10 20 30 Duration of sonication (min)

40

E c o CM CO

1.6

1.4

1.2

1 -

0.8

0.6

0.4

0.2

0 B

0

• 0.2M m 0.3M A0.5M • 0.6M

Diluted to indicated KCI concentration prior to sonication

10 20 Duration of sonication (min)

30 40

Fig. 4: Variations in turbidity of NAM sonicated under dissolved and suspended state.

(A) Changes in turbidity of sonicated NAM after diluting to 0.2, 0.3 and 0.5M by

Tris-maieate buffer (20mM, pH 7.1).

(B) Changes in turbidity of NAM sonicated under suspended state of 0.2, 0.3 and

0.5M KCI (in 20mM Tris-maleate; pH, 7.1).

NAM dissolved in 0.6M KCI (in 20mM Tris-maleate; pH, 7.1) was used as

control

29

60

50 -

^ 4 0 -

>% = 30 3

( ^ 2 0 -

10

0 A

y i

* -

• 0.1 M

J= y ^

/ ^

*

[ 1

• 0.2M

— A —•

0,3 M A

— •

-i !

60

50

5~40

I 30 I 20

10

0

5 10 20 30 Duration of ultrasonicalion(min)

• 0.1 M •0 .2M H O . S M

^ •

B 10 20 30

Duration of ultrasonication(min)

Fig. 5: (A) Effect on solubility of chicken NAM (4mg/ml) sonicated in dissolved state (0.6 M KCI with Tris-

maleate pH 7.1) and then diluted to 0.1, 0.2, and 0.3M with Tris-maleate (pH, 7.1).

(B) Effect on solubility of NAM (4mg/ml) sonicated in diluted state at 0.1, 0.2 and 0.3M with Tris-

maleate (pH, 7.1).

kD(Mr)

200

110 ~

46 42 39 25 23 20

18

16 •

t = =̂ ^

B 100 0 100

<

CO

o

Electrophoretic Mobility (AU)

Fig. 6: (A) Electrophoretic (SDS-PAGE, 10%) profiles of NAM dissolved in 0.6M KCl.

Lane-1 is control / unsonicated NAM; Sample collected at 5 (Lane-2), 10 (Lane-3), 20 (Lane-4)

and 30 min (Lane-5) following sonication were loaded in amount of lO^g in each lane.

(B) Densitograms of the representative lanes to show the difference in peaks.

31

0.1 M Supernatant (Diluted after sonication)

200

110 46 39 25 23 20 18 16

— — — — — — — —

• • • • • • • j ^I^MIH^^^I ^^^^^^p^^ ^̂ ^̂ 1̂ ^^^B ^^H ^̂ ^̂ _̂ m̂ ^^^^^B' ^^^^K ^H m̂ ^̂ l

1 2 4 5

0.1 M Supernatant (Suspended before sonication)

Fig. 7: (A) SDS-PAGE (10%) of sonicated NAM (0.6M KCI) diluted to O.IM after sonication. Equal quantity

of NAM (lOjig) was loaded in each well. Lane-I is control NAM (unsonicated); lane-2 is

supernatant of the Control; lanes 3-6 supematants of NAM sonicated for 5, 10, 20 and 30 min

respectively.

(B) SDS-PAGE (10%) of NAM diluted to 0.1 M first and then sonicated. Equal quantity of NAM

(lOjig) was loaded in each well. Lane-I is control NAM (unsonicated); lane-2 is supernatant of the

Control; lanes 3-6 supematants of NAM sonicated for 5, 10, 20 and 30 min respectively.

32

kD ( Mr̂ 200.

110—

Supernatant 0.2IVI

Pellet 0.2 M

Fig. 8: Electrophoretic (SDS-PAGE, 10%) profiles of NAM ultrasonicated in dissolved state (0.6 M KCI

(with 20mM Tris-maleate; pH, 7.1) at 20KHz for 30 min. Aliquots collected at 5, 10, 20 and 30

min were diluted to 0.21VI

(A) Supernatant obtained after diluting to 0.2M KCI. Lane's sequence: Lane-1 is control

NAM (unsonicated); lane-2 is supernatant of the Control; lanes 3-6 supematants of NAM

sonicated for 5, 10, 20 and 30 min.

(B) Pellet obtained after diluting the samples whose supematants were analyzed in the above

photograph (A). Sequence of loading is exactly in its accordance. Equal quantity of NAM

(lOng) was loaded in each well.

33

kDCM;̂ 200-

Supernatant 0.2 M

2 3 4 5 6

Pellet 0.2 M

Fig 9: SDS-PAGE (10%) profiles of NAM that were diluted to 0.2M first and then ultrasonicated.

(A) Profiles of supernatant at 0.2M KCi :Lane-l in each panel is undiluted control NAM (unsonicated); iane-2 is the supernatant of the control; lanes 3-6 are supematants of NAM sonicated for 5, W, 20 and 30 min, obtained after diluting with KCI of the same concentration (0.2M).

(B) Profiles of pellet of the supernatant samples analyzed in the above top photograph (A). Sequence of the pellet is also the same as of corresponding lane of its supernatant. Equal quantity of NAM (lOng) was loaded in each well.

34

0.18 c F c (D

() Q.

*•— O

F -n m m i _ (U

QI

«*— o

0.16

0.14

0.12

0.1

U.08

0.06

0 04

2 0,02

• Ca2+-ATPase»Mg2*-ATPase * K*(EDTA)-ATPase

0 + 10 20 30

Duration of ultrasonication(min)

Effect of ultrasonication on Ca^^-ATPase (•), Mg^^-ATPase (•) and K*-( EDTA)-ATPase ( . )

activities of aged actomyosin sonicated after storage of 21 days in crushed ice.

Control values of 0.27, 0.18 and O.MuM of P/'/mg/min were obtained for Ca^\ Mĝ ^ and K*-

(EDTA)-ATPase respectively. Other conditions were the same except that no dilutions were made

and ATPase activities of sonicated samples at different timings were as such assayed for each of

the above activities.

35

3̂^

>̂ XI 3 o (/)

100

90

80

70

60 50

40

30

20

10

0 5 10 20 30

Duration of ultrasonication(min)

Fig. 11: Solubility profile of aged actomyosin (4mg/ml) sonicated following 21 days storage in

dissolved state (0.6 M KCl in 20mM Tris-maleate; pH, 7.1). Sonicated samples were

diluted to 0.1 M.

36

kD (Mr)

.^ ^m *H « n i->

w^

2 0 0 -

1 1 6 -110

4 6 -42 _ jy 2 S -23 ~

J55 20

Fig. 12: Electrophoretic (SDS-PAGE, 10%) profiles of supernatant and pellet collected from sonicated

aged actomyosin. Lane-1 is control NAM (unsonicated); lanes 2- 5 are NAM collected at 5, 10, 20

and 30 min of sonication (Described under Material & Methods) respectively. Equal volume of

lOng was loaded in each lane.

37

Supernatant Pellet

Electrophoretic Mobility (AU) Electrophoretic Mobility (AU)

Fig. 13: Densitograms of supematants and pellets of SDS-PAGE profiles (Fig. 12) of sonicated

aged NAM preparations. (Details are same as given in Fig. 12)

CfiapterS

(EHscussion

38

The contribution of myofibrillar proteins is more important in poultry muscle

because collagenous tissue in it is remarkably less in comparison with cattle muscle.

Therefore, in case of poultry meat contribution of collagen in tenderness-loss during

post-mortem storage and cooking characteristics is proportionately small.

Acceptability of muscle as food largely depends on tenderness (decrease in

toughening), which is determined by a series of complex processes, fhe processes

include both intrinsic and extrinsic factors. Whether it is extrinsic or intrinsic factor,

tenderness in both cases is determined by the degree of alteration of structural muscle

proteins and its associated proteins (Hopkins and Taylor, 2002). Myofibrillar proteins

make up bulk of the edible mass of muscle and are also major determinants of its

properties. Factors affecting myofibrillar proteins will ultimately influence the edible

muscle mass.

In recent years, application of ultrasonication, as a potential extrinsic factor

to tenderize meat is gaining increasing attention (Zayas and Gorbatow 1978:

Dolatowski 1989; Ahmad and Hasnain, 2013a). The association between

ultrasonication and meat tenderization, as evident from literature, is indirect since,

ultrasonication is supposed to be due to release of proteases from lysosomes and their

activation (Koohmaraie, 1988; Koohmaraie, 1996). Correlation and individual

contribution of ultrasonication and proteases towards the meat tenderization is not

very well demarcated. Reports which indicate either dissociation or fragmentation of

isolated myofibrillar proteins by ultrasonication are too old (Barany et al., 1963;

Zayas, and Gurbatow, 1978; Dolatowiski, 1988) and, therefore, demands repetition

with modified and effective protocols. One way would be to investigate the main

contractile complex, outside the myofibrils. The contractile complex, which again

39

constitutes bulk of myofibrillar contents, is extractable as natural actomyosin or

NAM. Therefore, in this study NAM from chicken breast muscle, Pectoralis major,

was used to study the effect of ultrasonication on biochemical and molecular

properties of NAM.

Since NAM complex exists in insoluble state when inside myofibrils, the

investigations were carried out on NAM under insoluble as well as soluble state. The

i-csuKs of this study reveal that the Ca'^-ATPase and Mg'^-ATPase decreases with the

ultrasonication by 20-25% and 30-35% respectively (Fig. 1-2). K'-(EDTA)-ATPase

activity shows lesser degree of decrease with ultrasonication (Fig. 3). These results of

various ATPase assays indicate that enzymatic activity of NAM declines with

ultrasonication. Ultrasonication in liquid medium creates many physical, mechanical

and chemical effects (McClement, 1995) which are strong enough to change the

properties of constituents present in medium. Proteins in NAM complex, when

exposed to ultrasonic radiation were supposed either to get dissociated or fragmented.

Changes in Mg'"^-ATPase reveal that the association of actin and myosin weakens

with the increasing duration of ultrasonication. Mg'^-ATPase levels indicate the

strength of actin myosin interaction (Torigai and Konno, 1996). Ca'^-ATPase activity,

on the other hand, demonstrates the integrity of myosin molecule (Benjakul et al.,

1977; Roura and Crupkin 1995), The decrease in both of these parameters, in any

case, will amount to neutralize the protection provided by actin when it is tightly

bound with myosin as part of NAM complex. Myosin will, therefore, be exposed to

increased structural alterations by ultrasonication, that may be a mere conformational

change or partial denaturation.

40

This is evident from the behavior of aged NAM preparations, since during

aging also actin-myosin interaction weakens and denaturation sets. As comparatively

displayed by Figs. 1-3 and 10, susceptibility of Ca-"'-ATPase, Mg-'"-ATPase and K -̂

(l-DTAj-ATPase activities of NAM to ultrasonication visibly increased with aging of

the preparation under ice storage. Aged actomyosin shows more rapid decline in

enzymatic activity as compared with fresh samples. Ca'"^- and Mg""^-ATPase activity

of aged actomyosin gets declined by weakening of actin and myosin interaction

(Arakaawa et al., 1976; Li et al., 2012).

SDS-PAGE profiles of the supernatant from fresh NAM shows increase in

dissociation of NAM complex with sonication without causing any fragmentation.

Our SDS-PAGE results are in agreement with the earlier reports (Lyng et al. 1997;

Ahmad and Hasnain 2013a). Aged NAM, however, shows two polypeptides of

molecular weights ~142kD, and ~129kD. In the literature, serine proteases are known

lo cause such fragmentation and well characterized proteolytic fragments

corresponding to ~142kD and ~129kD are heavy meromyosin (HMM) and myosin

rod. In addition, the fragments of molecular weights ~95kD and ~84kD were

observed in the aged NAM sample sonicated for 30 min (Fig. 12, Lane 5). In the

literature, myosin subfragment SI, and light meromyosin (LMM) are the fragments of

molecular weights corresponding to these values (Lowey et al., 1969).

Solubility of proteins is a complex function of the physico-chemical nature

of the proteins, which is determined by many factors. It appears that ultrasonication of

proteins unfolds the protein structure, which exposes their hydrophilic sites to binding

of water molecules and thereby their solubility (Morel et al., 2000). The results show

that the interaction between actin, myosin and other regulatory proteins of NAM

41

decreases with the increasing exposure to ultrasonication. The radiation effect exposes

the water interacting residues of the complex resulting in the increase of solubility in

low ionic medium. This solubility transition from high to low ionic medium by

ultrasonication was also reported by Ito et al. (2003). Solubility attains the maximum

value at 10 min of sonication in our study. Ultrasonication does not affect the

solubility of fresh preparations of NAM in 0.1 M saU solution but increases the

solubility of aged NAM at the same salt concentration many fold. Because of

weakening of actomyosin interaction ultrasonication causes more rapid dissociation of

aged NAM complex along with the fragmentation of myosin heavy chain. As extra

hydrophilic residues are exposed in aged actomyosin by ultrasonication so, solubilit>

is comparatively more. When all the hydrophilic residues of actomyosin get exposed

and no more hydrophilic residues are left to interact with water, there will be no

further increase in solubility (Hasnain et al., 1976; Hasnain and Ahmad, 2006).

Present investigation describes the effect of ultrasonication on

dissociation/fragmentation and denaturation along with other biochemical properties

of extracted NAM of Gallus gallus. As small changes in structural proteins cause

alteration in enzymatic activities so the protein quality is more sensitively determined

by enzymatic properties. Decline in enzymatic activity of NAM by sonication depicts

the extent of structural alteration occurring when ultrasonication is used in meat

tenderization. Ultrasonication, as a pure physical force, is being employed in meat

industry as a suitable extrinsic factor to tenderize meat or solubilize myofibrils. The

basic aspects of the tenderization process are not well understood. This investigation

for the first time has taken up the effect of ultrasonic radiation taking in to account the

differential solubilities of major myofibrillar components. The data presented here

42

shows that for more drastic changes by ultrasonic radiation, NAM complex has to be

in a soluble state.

Cfiapter 6

Summary

43

This study was aimed to investigate tiie effect of ultrasonication on broiler

Natural Actomyosin (NAM), the principal structural complex of myofibrillar proteins.

The main objective was to compare its behavior with that of intact myofibrils, where

endogenous proteases are reported to cause degradation of certain proteins. NAM was

ultrasonicated in dissolved as well as suspended or partially diluted state for different

lime intervals over a duration of 30 min. The effect on physico-chemical

characteristics was studied to monitor the fate of individual components of the NAM

complex subsequent to ultrasonication. The solubility, turbidity and enzymatic

property of actomyosin were chosen as the indices of characteristic changes. Whether

NAM aging influences the selected parameters was also investigated.

The results of this study indicate that ultrasonication initially causes some

dissociation of proteins present in actomyosin complex. Prolonged sonication appears

to bring about conformational changes leading to low-ionic-strength solubilization o\~

the proteins of NAM complex that in control were soluble at high ionic strength. At

the frequency (20 KHz) applied in this study, various proteins associated with this

complex are not fragmented or denatured in fresh NAM samples. The enzymatic

activity of fresh actomyosin complex decreases with ultrasonication, but the

solubility/insolubility of the complex is also important determinant. The solubility

increases up to 10 min of ultrasonication after that it remains constant. Turbidity also

decreases with ultrasonication showing an apparent dissociation or intra-molecular

conformational changes of actomyosin complex. Cleavage of myosin, as evident by

SDS-PAGE with remarkably increased solubility and inactivation of ATPase, occurs

only in aged actomyosin. The response of fresh as well as aged NAM to ultrasonic

44

radiation depends upon the duration of exposure, frequency of sonication and state ol

solubility.

Cfiapter 7

(BiBRograpfiy

45

Ahmad R, Hasnain A. (2013a). Ultrasonication of chicken natural actomyosin: Effect

on ATPase activity, turbidity and SDS-PAGE profiles at different protein

concentrations. Am J Biochem Mol Biol. DOI:10.3923/ajbmb.20I3.

Ahmad R, Hasnain A. (2013b). Peptide mapping of polymorphic myosin heavy chain

isoforms in different muscle types of some freshwater teleosts. Fish Physiol

Biochem. DOl 10.1007/sl0695-012-9735-9..

Ahmad R, Hasnain A. (2006). Correlation between biochemical properties and

adaptive diversity of skeletal muscle myofibrils and myosin of some air-

breathing teleost. Ind J Biochem Biophys. 43:217-225.

Arakawa N, Inagaki C, Kitamura T, Fujiki S, Fujimaki M. (1976). Some possible

evidences for an alteration in the actin-myosin interaction in stored muscles.

Agricultur Biol Chem. 40(7);1445-1447.

Asakura S. (1961). F-actin adenosine triphosphatase activated under sonic vibration.

Biochim Biophys Acta. 52(l):65-75.

Asghar A, Samejima K, Yasui T. (1985). Functionality of muscle proteins in gelation

mechanisms of structured meat products. CRC Crit Rev Food Sci Nulr.

22:27-30.

Babji AS, Kee GS. (1994). Changes in sarcoplasmic and myofibrillar proteins of

Spent Hen and Broiler meat during the processessing of Surimi-like material

(Ayami). Pertanika J Trap Agric Sci. 17(2): 117-123.

46

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>J ;̂̂ \̂ ^ âdt,J

Ac^jiow&djgements

Jit the very onset, I ta^ this opportunity to ej(press with profoundgratitude

and highest veneration, and sincere than^ to my revered mentor, (Dr. 'lijaz JLhmad,

^Assistant (professor, (Dept. ofZoolbgy, JiCigarh MusCim University, for his esteemed

guidance, practicality, ^en interest and incessant encouragement. I remain sincere fy

oSCiged to him for his trust, wisdom and exceptional cooperation at aCC times. His

encouragement and quality guidance provided the good Basis for fending me

innovative and creative thin^ng.

Jl great deSt is owed to (Prof. Irfan Afimad, Chairman, (Department of Zoology,

JiMV, for providing me all the facilities to pursue my research to its successful

completion.

I deeply acknowledge (Dr. ^Bsar-udHasnainfor his valuaSle and sustained

help, principled approach, prudent suggestions and constant encouragement during

the entire duration of this wor^

I would li^ to than^(Prof, Iq6aC(Parwez for continual encouragement, help

and constructive suggestions. Ji note ofthan^ is also due for (Prof. S^MJi A^idi to

expend ultrasonication and other facilities for my research wor^

It was always inspirational to have very supportive lab colleagues with

whom; it was a pleasure to wor^ A to^n of appreciation will always remain for

Ms. JireeSa Ahmad for her everlasting cooperation and sincere support in the lab.

I am fortunate enough to have Itnran, IqSal, Sajjad, Lateef, ^Nazia,

SiBgatuQafi, ^Nuzhat andYusra as my research colleagues. I owe them a lot for their

timely help and discussions. I appreciate the care, unreserved support, cooperation

and motivation I received from tKasftim and^^ffi throughout my stay injiligarh.

I would li^ to than^yfannanfor ta^ng his time out for helping me during

sonication process. I shall always recall the help expended By Or. iNiisim Jihmad,

'Young Scientist,

I l l

WitH aCC sincerity, I convey my gratitude towarcCs my revered parents. Their

exquisite Benedictions, unflinching support, good wishes. Cove and affection right

from the day of my advent to this earthly ivorCd6ecame my strength that inspired me

to achieve success hi the academic worCdandaSove aCCfuCfiCCedmy wishes.

TinaCCy finahsiaC assistance from V^C is aCso ac^owCedged as pari of the

wor^was carried from th£ financial assistance of the 'S^search

IV

APPENDIX

1. Solutions for ATPase assays

(i) Ammonium moiybdate (2.5%)

1.25gm of Ammonium moiybdate + 4.06ml H2SO4. Final volume

raise to 50ml by distilled water.

(ii) ANSA (0.25%)

0.0625gm of ANSA + 24.37ml of Sodium bisulfate + 0.625ml of

Sodium sulfite.

2. Solutions for SDS-PAGE

(i) Upper Tris

Tris Base, 0.125M adjusted to pH 6.8 with HCl

(ii) Lower Tris

Tris Base, 0.375M adjusted to pH 8.8 with HCl

(iii) Acrylamide stock (30%)

29.2gm of Acrylamide + 0.8gm of Bis-acrylamide dissolve in

100ml of water

(iv) APS stock (10%)

O.lgmofAPSin 1ml.

(v) Sample Buffer stock (4X)

0.30ml of Tris (0.25M) + 4ml of Glycerol + 2ml of 2-

mercaptoethanol + 0.0004gm of Bromophenol blue + 0.8gm SDS.

Dissolve in 10ml of distilled water.

Sample preparation for SDS-PAGE

Samples were prepared by mixing protein (NAM) sample and 4X

sample buffer (pH 6.8, 10% SDS, 2mM 2-mercaptoetiianol, 20%

glycerol, 0.001% w/v BPB) in the ration of 3:1. Samples were kept in

boiling water batii for 5 min.

SDS-PA gels preparations

The gels contained 10% acrylamide (acrylamide 30%: bis-acrylamide

0.8%) and \0% glycerol. Just before polymerizing the gels, ammonium

persulfate (10%) and TEMED were added to final concentration of

0.3% and 0.03-0.05%, respectively.

Preparation of CBB-R250

225 ml of Methanol (45% v/v) + 45ml Acetic acid + 1.25gm of CBB-

R250. Raise the volume to 500 ml by distilled water.