Osterhoff, D.R.

Pieterse, G

Ward-Cox, I.S.

English

AJOL - African Journals Online

S. Afr .  J.  Anim. Scr.  I ,103 -107 (197|)PRELIMINARY RESULTS ON ELECTROPHORETIC ANDIMMUNOELECTROPHORETIC FRACTIONATION OFBOVINE MUSCLE EXTRACTD.R. Osterhoff. I.S. Ward-Cox and G. PieterseDeryrtment of Zootechnologt, Facult, of Veterinary Science, OnderstepoortOPSOMMING: VOORLOPICE RESULTATE VAN ELEKTROFORETIESE EN IMMUNOELEKTROFORETIESE SKEIDING VAN BEESSPIEREKSTRAK.Besonderhede van teglieke van styselgel-, sellulose asetaat- en immunoelektroforese is gegee, wat 'n skeiding van spierekstrrk op so 'n ma-nier moontlik maak, dat variasies tussen individuele monste$ kwalitatief opg€spoor kan word. Die reproduseetbaarheid van resultate by stysel-gel- en sellulose-asetaat elektroforese was besonder goed tcrwyl klein variasies in die tegniek sekeae analieses by immunoelektroforese bemoeilikhet. Maa-r ook hier kom later 'n goeie teproduseerbaarheid yan resultate teweeggebring word. Die volgende stap in hierdie studie is die bepslingvan die oorerwing van die fraksies wat deur die drie tegnieke geskei kon word, en die ondeBoek van spierprolein veranderinge in kalwe$ metspastiese parese.SUMMARYDetails of techniques aae presented of starch gpl-, cellulos€ acetate - and immunoelecttophoresis which can be used to fractionate bovinemuscle extract in such a way that the variation between individual samples can be detected qualitatively. The rcpeatability of results in starchgeF and cellulose acetate electrcphoresis was very good whereassmall variationsin the technique of the immunoelectrophoresis hindered correctanalysis. Later, however, cle&r repestability of results could also be obtained here. The following step in this study will be the determination ofthe hereditaly backgtound of these fr.ctions s€palated by the three techniques. and the irvestigatior ol mwcle paotein changes in calves withspastic parcsis.hocedure, Specimens were collected from the following muscles:M. gastrocnemius, M. biceps femoris and M. brachio-ceplnlicus, the first two being those affected by spasticparesis. Spastic paresis is a deleterious trait with a geneticbackground which is causing increasing concern in certaincattle breeds. The muscle specimens were obtained from72normal animals whilst they were still on the hook in theprocess of being skinned, i.e. pre-rigor muscle samples.Being aware of the relatively quick changes in pH, lactatesand energy-rich phosphates small pieces of approximately20 g were dissected out from the three muscles and treatedimmediately in the following way: after transporting thesamples in phosphate buffered saline to the laboratory - adistance of only 300 meters - the specimens were cut intofine pieces and swung down in an ultra-centrifuge at100 000 g, according to the technique of Kossmann ( 1969).This process was also carried out as soon as possible so as toavoid excess denaturation and consequent pH changes(Scopes & Lawrie, 1963). After 30 mins. the samples wereremoved and the supernatant (approximately 4 ml forevery l4 g meat) was decanted and either processed imme-diately or stored at -l5oC for use the following day.Starch gel electrophoresis was carried out using theprocedure of Kossmann but with the following buffers: forthe gels, 3,6 g Trishydroxymethylaminomethane and 0,8 gcitric acid made up to I l i tre with disti l led water, and forthe electrodes, l 2 g LiOH and I 1,8 g Boric acid made upto I litre with distilled water. Using 24 gConnaught hydro-lysed starch in 186 ml gel buffer, the gels were cast in pers-pex molds measuring 24 cm x 1 5,5 cm x 3 mm. A current of400 volts and 50 mA was applied unti l the borate boundaryhad nrigrated ca. l0 cm, after a period of ca. 3 hours. CareThe first electrophoretic studies on the proteins ofmuscle tissue were carried out by Bosch (1951), who foundthat differences existed between muscles from differentanimals as well as between muscles from the same animal.Hughes (1959, 1960) performed similar studies on humanmuscle tissue and identified different myoglobin andmyoalbumin fractions. He found that quantitative diffe-rences between these two fractions were related to muscleatrophy due to denervation. Kronman, Weinberger &Winterbottom (1960) investigated the biochemical andelectrophoretic differences between eight different bovinemuscles and found that they differed with respect towater soluble proteins, aldolase and ultimate pH. Quanti-tative differences also existed amongst the various proteinfractions of different muscles together with variations inthe electrophoretic patterns. Kossmann (1969) found greatindividual differences in the esterase in his pherograms ofproteins of bovine skeletal musculature and was able toestablish genetic polymorphism for two esterase systems.From a phylogenetic point of view, Giles (1962) investigated the differences between the sarcoplasmic proteinsof beef, mutton and pork, and demonstrated a similarpattern for ruminants in contrast to the pig. Similar workhas been done in sea vertebrates, namely by Tsuyki,Roberts & Gadd (1962) on the Pacific salmon and byHapner, Bradshaw, Hartzell & Gurd (1968) on the seal,porpoise and whale, using electrophoretic procedures.Their findings, like those of Hartshorne and Perry (1962)revealed distinct phylogenetic as well as ontogenetic dif-ferences in the sarcoplasm. The object of the present studywas to extend the practical applications of biochemicalgenetic typing to an investigation of muscle proteins fromcattle.103was exercised in keeping the gels co<ll during the process asexcess heat tended to give illegible results. On completionof the run, each gel was removed from its tray and stainedwith amidoschwarz l0B. the patterns being read on theunderside.Zone electrophoresis was also done on celluloseacetate membranes so as to obtain clear separation of themyoglobins. In the work of Kossmann (1969) no variationcould be found in this fraetion. Using a buffer composedof 6,2 g Trishydroxymethylaminomethane, 1,6 E citricacid made up to I 000 ml with distilled water, and a cur-rent of l66V and 4 mA, the membranes were stained withbenzidine after a running time of 1l/z hours.lmmunoelectrophoresis was performed using themicro-technique of Grabar and Burtin (1964), and a conti-nuous barbitone-acetate buffer system, ionicity 0,1; pH 8,6.Electrophoresis was carried out at 250 volts and 25 mA for2 hours. Precipitation was accomplished with rabbit anti-bovine serum (RAB 20) and a pooled antiserum, the mini-mum reaction time being 16 hours. Excess antigen tookabout 48 h to dialyse out sufficiently to give a clear back-ground, presumably due to the high protein and fat con'tent of the extract.For the production of suitable precipitating antisera,it was decided to alter the technique as applied by Ward-Cox (1971) as follows: meat juice was injected into fiverabbits in 5 ml aliquots weekly for two weeks followed byalum-precipitated serum globulin on the third week andwhole serum on the fifth week. On the sixth week theywere bled, the antibodies having been controlled on im-munodiffusion plates. After a further immunoelectro-phoretic control, making use of the strongest antisera on-ly, these were pooled to form the final product.Discussion of ResultsZone electrophoresis in starch gel: From Fig. I it is evidentthat the nrain fractions along the migration route (the in-sertion line is indicated on the lower side of the photo-graph) fall into three clear categories, appearing as themore intensely coloured bands on the electropherogramand marked, in order of increasing migration rate, Mg, Mband Ma, Mg is clearly the myoglobin fraction of Hughes(1959) due to its peroxidase activity as revealed by stainingwith benzidine. Using a bovine serum control, it was fur-ther ascertained that the fastest band Ma corresponded tothe albumin, also in agreement with Hughes. The zone Mbcould not be differentiated, but from the immunoelectro-phoretic patterns it appears that it is closely related to theserum transferrins. However this was not verified by makinguse of Fe-binding estimates. It can be seen that this zonemigrates, somewhat faster than the serum transferrins, andunlike the transferrins, it shows no variability from one in-dividual to another, using this specific technique. The onlyvariation that was observed occurred in the Mc-zone migra-ting midway between the Mg-zone and the Mb-zone, butwhich was still slower than the serum transferrins. Thisis also in agreenrent with Kossmann (1969). However, hefound only the two types which could be postulated asbeing the heterozygote and the faster-moving homozygote,whereas this investigation revealed the second hymozygoteas well. For the sake of convenience they are termed McAA,McBB and MoAB. The actual results obtained by starchgel electrophoresis are presented in Table l.Table IClossification of investigated muscle samplesPercentage of different typesMcAA McBB McABM, gostrocnemiusM. biceps femorisM. brachio-cephalicus59,150,034,522,' l1 5 , 43 1 , 0r 8.234,634,5MaMb* rrr; ; J-M ff iM* #m#Wail#.qt q € F * %u& p*{ffi ffi -'M cn +6 !'li 't***+ffi MgffiFBB AB AA BB F ot BB AA BB AB BB Mc-typesFig. t . - Starch Sri pnnogrom of bovine muscle extractThe actual importance of these different types hasnot yet been investigated, and await genetic studies toprove whether another polymorphic system is heritable.Zone electrophoresis on cellulose acetate: Myoglobin typingusing this technique revealed two distinct types, vu. asingle banded and a double banded formation, whichshowed a variation (see Fig. 2). One specimen gavea slower moving band which may have been due todenaturation and can thus not be considered at thisstage. There appeared to be no qualitative variation be-tween muscles of the same animal but clear quantitativedifferences were obvious as in Fig. 3, where it can be seenthat the M. biceps femoris showed more intense myoglobinbands and the M. brachio-ceplwlicus the least intensity.to4l3G32G33Gl6G3 lG2rc33GlsGFg. 2. - Myoglobin types of M. Gastocnemius in differentanirnalsThe results of the actual typing showed that in 80,6%of all samples of M. gastrocnemius, the myoglobin appearedin two bands, while 19,4% showed only one band.In the samples of M. biceps 88,9 % possessed two bands andonly Il,l% a single band, while the myoglobin concen-tration in the M. brachio-cepholicus was too weak toenable clear separation.Agar gel immunoelectrophoresis: A variety of fractions ex-hibit immune patterns in this technique, as can be seenfrom Fig. 4 where an attempt has been made to depict allthe precipitin arcs that were observed. There were threemajor arcs, as was also the case in the starch gel patternswhere three major zones could be seen. On the basis oftheir migration properties as compared to the starch gelpatterns, the terms Ma, Mg and Mc were also used. Thearcs Mg and Mc appear as either single or double forma-tions. The variation of the Mc are coincided with thatof its counterpart on starch gel although it was not pos-sible at this stage to distinguish between the homo- andheterozygotes by this technique. The variations of theMg arc cannot, however, be correlated with any similarphenomenon on starch gel. The small differences thatKossman considered too insignificant to take into accountmay be explained by this method, as he carried out spe-cific tests on a variety of myoglobin samples.When compared with the immunoelectrophoreticpattern obtained with whole serum, as in Fig. 5, remark-able similarities occurred. Although the original sampleswere taken into PBS (phosphate buffered saline) and tho-roughly washed in this solution before processing, it wastherefore impossible to set a clear differentiation betweenmuscle and vascular protein fractions although there manydifferences exist. Where serum (b) showed up to thirty-twoarcs, the muscle proteins (a) showed only a maximum often, including the variations of the Mg and the Mc arcs. Ge-nerally speaking, the arcs of serum protein fractions weremore extensive than those of meat extract. This was mostobvious in the albumin, transferrin (siderophilin) andy-globulin arcs. Certain major serum protein arcs werecompletely lacking in meat extract, e.g. the 02 -macro-globulin (c) which had its origin in the antigen well and ex-tended towards ttre albumin arc.In accordance with the general patterns obtainedwith starch and agar, the major variations occurred withinthe three main arcs, as illustrated in Fig. 6. There werethree combinations in the samples to be found, viz. theMa arc alone (28B), the Ma and the Mg arcs ( l9 BC, 31 G,39 B), and the Ma, Mc and Mg arcs (62G, 38 B). More arcscould actually be seen but these were the clearest onesfound in the extracts of the three muscles used. Further-more, these types were distributed amongst all the musclesbut detailed genetic analysis has still to be carried out. Itwas noticeable that the Ma arc was always present but theMg and the Mc arcs were absent in many instances. Thequantitative differences previously mentioned were verypronounced in the Ma arcs but not in the others.This fact has been observed in all the material investisatedand is shown in Fie. 7.iltffiill9Gl l Gt$- =  - - '3 l BFg. 3. - Myoglobin types of tlvee differenttwo onimols32G3 l G, . : t  j : ,tf;eSt: a ; : 1328cmuscles in105Mo\irxrili.'"rr.*-"-.Frg. 5. - Comparison o| immunoelectophoretic Wtterns ol'(a) muscle extract, (b) blood serum and (c) a2-mauoglobulin288l9BCVarious combirutions ol'three moior immuno-electrophoretic fractions of muscle extrqct aftera running time of I I 12 hoursFrg. 7. - variation o|the immunoelectophoretic liactionsin ntto muscles of the some animal ofter o runningtime of 2 hoursThe difference in the mobility between the Mg arcsof two muscles from one animal, as in slide l3K of Fig.7,although not observed in blood senrm using the sametechnical procedure, ffiay be attributed to the same pheno-menon as observed by Nansen ( 1970). using blood serumhe has classified immunoglobulins in terms of electrophoreticmobility as being IgG fast and IgG slow. This nomenclaturehas not been finally established. However, in spite of thefact that the denaturation of muscre proteins was an uncon-trolled factor in this specific type of work, good repeatabi-lity of results was obtained in all three techniques. The im-mediate treatment of the pre-rigor muscle samples apparent-ly helped to overcome the initial difficulties expiiiencedespecially in the immunoelectrophoretic technique. Sum-marising the significance of these findings one could men-tion the following practical applications:a. From anatomical studies performed at onderste-poort it appears that M. biceps femoris and espe-cially M. gastrocnemius are involved in the abnor_mal muscle development in cases of spastic paresisMFig. 4. - Diagrom of immunoelectrophoretic fractions of muscle extractM9"tiln62BCpooled antircnrm52G65 BCpoohd antiserum6 5 C65 BCRAB 206 5 G6EGpoolcd antircnrm68 BC(b)(c)Fig. 6. -106b.in calves. This disease expresses itself in an uncon'trolled stiffness of one or both hindlegs and is apparently inherited via an autosomal semi-lethalrecessive gene. The possibility that the heterozygouscarriers of this trait could be established by takingsamples from suspected living carriers with abiopsy needle needs to be investigated.Using the techniques described a further objectivedetermination of some of these qualities of meatwhich are difficult to measure,could be established,e.g. colour, juiciness and texture. The next stepwould then be a correlation of the clearly esta-blished variants of muscle extracts with thesequality characteristics. It must be borne in mind,however that the sarcoplasmic proteins are labileand readily denatured under differing pH and tem-perature conditions. The next step would then bean investigation of the hereditary backgroundof the established types as mentioned above.There remains the posibility of using thesetechniques in the more remote fields of immu-nogenetics, namely phylogenetic systematics andbiosystematics. Until recently, immunogeneticistshave restricted their pool of genetic markers tothose on the red and white blood cells and to theserum proteins and enzymes. If, for example, themyoglobins, could be included in these systematicclassifications breed relationships and species clas-sifications could be performed on an even broaderbasis.AcknowledgementThe authors wish to thank Miss V.W. Emslie of theTrypanosamiasis Research Unit, Dept. of Veterinary Re-search, Salisbury, for her invaluable contribution to thisproject.ReferencesBOSCH,  M.W. ,  1951.  B ioch im.  b iophys .  Ac ta  '1 ,61 .GILES, B.G.,  1962. J.  Sci .  Fd. Agr ic.  13,164.GRABAR, P. & BURTIN, P., 1964.lmmunoelectrophoreticAnalysis. Amsterdam: Elsevier.TIAPNER, K.D., BRADSHAW, R.A., HARTZELL, C.R. &GURD, F.R.N.,  1968, J.  b io l .  Chem. 243,283.HARTSHORNE, D.J. & PERRY, S.V., 1962. Biochem.J .  8 5 ,  l 7 l .HUGHES, 8.P., 1959. Biochem. biophys. Res. Commun.l ,  194.HUGHES, B.P.,  1960. Nature,Lond.,  188, 1028.KOSSMANN, H., 1969. Untersuchung von Proteinen derSkelett-muskulatur des Rindes mit Hilfe der Stdrke-gelelektrophorese. Inaug. Diss. Giittingen.KRONMANN, M.J., WEINBERGER, L.E. & WINTER-BOTTOM, R.J., 1960. Archs Biochem. Biophys.86, 238.NANSEN. P.,1970. Metabolism of Bovine Immunoglobulin -G.- G.A Clinical and Pathophysiological study. Copen-hagen: Munksgaard.SCOPES, R.K. & LAWRIE, R.A., 1963. Nature, Lond.t97, t202.TSUYUKI, H., ROBERTS, E. & GADD, R.E.A., 1962.Can. J. Biochem. Physiol. 40,929.WARD-COX, I.S., lg7l.Jl S.Afr. vet. med. Ass. 42, 185 .c.107

Preliminary results on electrophoretic and immunoelectrophoretic fractionation of bovine muscle extract

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Preliminary results on electrophoretic and immunoelectrophoretic fractionation of bovine muscle extract

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