p. 571 to 572. Pergamon Press. Printed in (
~SIS OF THE HIGH-TYR ON CELLULOSE ACET
)ROTEINS 7,IPS
ROVE, M. J. FRENKEL AND J. M. G1 3.S.I.R.O., Parkville (Melbourne), Victol
ralia
(Received 7 January 1974)
ABstragt--l. A metnoo is oescrloeO ~ed for Ior the electr( electropnoreslS )horesis of o! the tl high-tyn cellulose acetate which provides resolution comparable with th the previous method uses much less protein (5 vs. 50 pg), is much quicker quicl (1.5 hr sharper and cleaner patterns than the starch gel procedure. 2. The staining of proteins on cellulose acetate with Coomassie Coon~ brillian the introduction of a one bath fixing and staining procedure tel)gether with This now means that the fiftyfold increase in sensitivity of Coomassie Co( bh used Ponceau S stain can be achieved more easily.
INTRODUCTION rALIAN keratins contain three main groups of is, low-sulphur, high-sulphur and high-tyro•oteins, which can be obtained in solution as oxymethyl kerateines by alkaline reduction of S-carbox the parent a'ent keratin followed by alkylation of the soluble proteins with iodoacetate. All three protein types are quite heterogeneous and zone electrophoresis is has been extensively used to investigate the degree of this heterogeneity. Most;t zone electrophoretic studies of keratin hightyrosine he proteins have been carried out in starch gels at alkaline aline pH, but this is a lengthy procedure requiring ng at least a day from gel preparation to the completion of destaining. This limits the use of starch in following chromatogra~phic fractionations where it is often desirable to know mow the degree of tion does not apply separation quickly. This limitation 'esis which requires to cellulose acetate electrophoresis only 1-2 hr for each experiment. Although cellulose acetate is now widely used [but not as yet for the keratin proteins], it may not be suitable for the routine examination of all protein in systems. Gilliam & Valentine (1973) were unable ble to successfully electrophorese haemolymph proteins )teins of the honeybee on cellulose acetate strips. It should be pointed out thatt electrophoresis on :r of discriminating cellulose acetate lacks the power molecules of different sizes, a characteristic of starch. Therefore the resolution! of both these procedures will only be comparable; in systems like the high-tyrosine proteins wliere s~e ;ize differences are minimal, or the high-sulphur proteins )reins at at alkaline pH values where the relation between ~en size and charge tends to reduce resolution in sievm ;ving media.
of keratins on ure. The new ys) and gives 1 improved by "or destaining. he commonly
This pa paper descri ~ssful use of electroresolving the highphoresis on ( cellulo proteins fi ~atins, wool, porcutyrosine I hairs, and the pine quill, quil! guinea pig and mouse ha mot proteins, chromateagraphic fractions of the mouse with resolution resc comparable to that obtained in els. It is hoped that subsequenl uent studies can starch gel extend the th( use of electrophoresis in celhalose acetate to other keratin proteins. MATERIALS AND METHOD:s The prel preparation of Type I high-tyrosine proteins from four four kerat~ keratins, their electrophoresis in starch gels at pH 8.6 and the the chromatographic ehl and resolution of the rrrouse proteins on QAE-cellulose were carried out by the th( methods of Gillespie (1972a, b). Electrophoresis on cellulose acetate strips str was performed using a Beckman Microzone Electrophoresis El system. The buffer contained barbitone (0.09 (0.( M), NaOH pies (0-25/~1of a (0.075M) and urea (8 M) at pH 8.9. Sample 2% protein solution) were loaded with the t] applicator supplied with the instrument, and electro~phoresis was carried out for 60 rain at 250 V with aa constant const current of 3 mA. (When less concentrated solutions are available, e.g. chromatographic eluants, as many, as fo~ four loadings of 0.25 al can be made on a single position wit ition without appreciable distortion of the patterns.) The membranes mer were stained for 2-3 min with 0.2% Coomassie brilliant blue G 250 (I.C.I.) in water-methanol-acetic etic aci acid (45 : 45 : l0 v/v) as described for sodium dodecyl sulphate sulpha acrylamide electrophoresis by Weber & Osborn (1969), (196 and then -methanol-acetic acid destained for about 5 rain in water-methan, (60 : 30 : l 0 v/v). Constant agitation was necessary for rapid, even staining and destaining. The membranes were stored and photographed in 10% acetic aceti( acid. When the membranes were to be scanned the procedure of Fazekas et al. (1963) was used. A fex~ few membranes
571
AGROVE) M . J. FRENKEL AND J. M . GILLI/
uffers, and these was impossible, take of buffer, ON experiments in were run on e acetate at pH e Type I high1 into a similar . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . rid . . . .on . . . cellulose . . . . . . . . ,~, but the oands on the latter are sharper, defined and there is less background staining, correspondence in mobilities is not expected e some sieving and non-specific interaction occur in the starch system. Similarly, when ring the chromatographic fractions of the hair high-tyrosine proteins (Fig. 2), it can be hat cellulose acetate gives resolution come to starch but with greatly increased clarity patterns especially where minor components lcerned. significant that starch gel runs involve a 1 hr period or overnight when urea is present, r electrophoresis and 3 hr for staining, whereas se acetate involves a total time of only about There is also a major difference in the amounttt of protein loaded, with about 5/zg being 3r cellulose acetate compared with 50/zg for used for starch gels. The cellulose acetate technique described above retains the simplicity of single baths for staining and dng which is a feature of the widely used destaini~ m S method. The separate fixing step and Ponceau le destaining baths of Fazekas et aL (1963) multiple have been found unnecessary. Coomassie blue, er, has the advantage :y (Fazekas (i however, g( of its sensitivity et al., 1963) which we estimate to3 be some fifty times greater than Ponceau S. he cellulose acetate We have successfully used the electrophoresis procedure at IpH values ranging
from 2-4 to 12 wit resolution of both and II). In contras membranes of ot Valentine, 1973), v Millipore membral handle wet or dry. in the degree of r( batches of membra the high-tyrosine pJ the storage globulil angustifoh [(Lupinus LltplnUS Electrc Electrophoresis method ccurrently tyrosine ]proteins c applicatk )lication and th of the Cc Coomassie [ proceedir to imp1 proceeding nique to (other keta
ut 8 M urea for the ~-tyrosine proteins (I nt report relating to cturers (Gilliam & d that the Beckman nly and are easy to ty has been observed ained with different ~roteins as diverse as ns from keratins and eed of the blue lupin acetate is the best examining the high;cause of its ease of tivity and simplicity procedure. Work is ication of this tech-
s R. G. & DAYTNERA. (1963)Two new s dures for quantitative estimati estimation of pl lectrophoretic strips. Biochim. 91. Biochim biophys, z 1-fOtglllb; rich I1~I1 in 11 glycine and J. 1¥1. M. ~lYl/,,ii) (1~ GILLESPIEd. rrosine from keratins. Comp. Biochem. PhysioL 41B, tyrosine 723-734. aatemary ammonium ]' I LLESPIEJ. M. ( 1 9 7 2 b ) The use of quaterna GILLESPIE in the fractionation ot of tyrosine-rich ethyl cellulose c( proteins )roteins from wool. J. Chromatogr. 72, 319-324. ! GILLIAMl~ Jnusual electroM. & VALENTINED. K. (1973) UI phoretk )horetic behaviour of proteins from honey hol bee hemolymph. Comp. Biochem. Physiol. 45B, 4( 463-466. reliability of molecuWEBERK. & OSBORNM. (1969) The reliabi lar wei~ght determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406--4412. E ST. GROq FAZEKAS DE
Key Word Index--Mammalian keratins ; high-tyrosine porcupine quill; proteins; mouse hair; guinea pig hair; p( merino wool; cellulose acetate; Coomassie brilliant blue.
~SIS OF THE HIGH-TYR ON CELLULOSE ACET
)ROTEINS 7,IPS
ROVE, M. J. FRENKEL AND J. M. G1 3.S.I.R.O., Parkville (Melbourne), Victol
ralia
(Received 7 January 1974)
ABstragt--l. A metnoo is oescrloeO ~ed for Ior the electr( electropnoreslS )horesis of o! the tl high-tyn cellulose acetate which provides resolution comparable with th the previous method uses much less protein (5 vs. 50 pg), is much quicker quicl (1.5 hr sharper and cleaner patterns than the starch gel procedure. 2. The staining of proteins on cellulose acetate with Coomassie Coon~ brillian the introduction of a one bath fixing and staining procedure tel)gether with This now means that the fiftyfold increase in sensitivity of Coomassie Co( bh used Ponceau S stain can be achieved more easily.
INTRODUCTION rALIAN keratins contain three main groups of is, low-sulphur, high-sulphur and high-tyro•oteins, which can be obtained in solution as oxymethyl kerateines by alkaline reduction of S-carbox the parent a'ent keratin followed by alkylation of the soluble proteins with iodoacetate. All three protein types are quite heterogeneous and zone electrophoresis is has been extensively used to investigate the degree of this heterogeneity. Most;t zone electrophoretic studies of keratin hightyrosine he proteins have been carried out in starch gels at alkaline aline pH, but this is a lengthy procedure requiring ng at least a day from gel preparation to the completion of destaining. This limits the use of starch in following chromatogra~phic fractionations where it is often desirable to know mow the degree of tion does not apply separation quickly. This limitation 'esis which requires to cellulose acetate electrophoresis only 1-2 hr for each experiment. Although cellulose acetate is now widely used [but not as yet for the keratin proteins], it may not be suitable for the routine examination of all protein in systems. Gilliam & Valentine (1973) were unable ble to successfully electrophorese haemolymph proteins )teins of the honeybee on cellulose acetate strips. It should be pointed out thatt electrophoresis on :r of discriminating cellulose acetate lacks the power molecules of different sizes, a characteristic of starch. Therefore the resolution! of both these procedures will only be comparable; in systems like the high-tyrosine proteins wliere s~e ;ize differences are minimal, or the high-sulphur proteins )reins at at alkaline pH values where the relation between ~en size and charge tends to reduce resolution in sievm ;ving media.
of keratins on ure. The new ys) and gives 1 improved by "or destaining. he commonly
This pa paper descri ~ssful use of electroresolving the highphoresis on ( cellulo proteins fi ~atins, wool, porcutyrosine I hairs, and the pine quill, quil! guinea pig and mouse ha mot proteins, chromateagraphic fractions of the mouse with resolution resc comparable to that obtained in els. It is hoped that subsequenl uent studies can starch gel extend the th( use of electrophoresis in celhalose acetate to other keratin proteins. MATERIALS AND METHOD:s The prel preparation of Type I high-tyrosine proteins from four four kerat~ keratins, their electrophoresis in starch gels at pH 8.6 and the the chromatographic ehl and resolution of the rrrouse proteins on QAE-cellulose were carried out by the th( methods of Gillespie (1972a, b). Electrophoresis on cellulose acetate strips str was performed using a Beckman Microzone Electrophoresis El system. The buffer contained barbitone (0.09 (0.( M), NaOH pies (0-25/~1of a (0.075M) and urea (8 M) at pH 8.9. Sample 2% protein solution) were loaded with the t] applicator supplied with the instrument, and electro~phoresis was carried out for 60 rain at 250 V with aa constant const current of 3 mA. (When less concentrated solutions are available, e.g. chromatographic eluants, as many, as fo~ four loadings of 0.25 al can be made on a single position wit ition without appreciable distortion of the patterns.) The membranes mer were stained for 2-3 min with 0.2% Coomassie brilliant blue G 250 (I.C.I.) in water-methanol-acetic etic aci acid (45 : 45 : l0 v/v) as described for sodium dodecyl sulphate sulpha acrylamide electrophoresis by Weber & Osborn (1969), (196 and then -methanol-acetic acid destained for about 5 rain in water-methan, (60 : 30 : l 0 v/v). Constant agitation was necessary for rapid, even staining and destaining. The membranes were stored and photographed in 10% acetic aceti( acid. When the membranes were to be scanned the procedure of Fazekas et al. (1963) was used. A fex~ few membranes
571
AGROVE) M . J. FRENKEL AND J. M . GILLI/
uffers, and these was impossible, take of buffer, ON experiments in were run on e acetate at pH e Type I high1 into a similar . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . rid . . . .on . . . cellulose . . . . . . . . ,~, but the oands on the latter are sharper, defined and there is less background staining, correspondence in mobilities is not expected e some sieving and non-specific interaction occur in the starch system. Similarly, when ring the chromatographic fractions of the hair high-tyrosine proteins (Fig. 2), it can be hat cellulose acetate gives resolution come to starch but with greatly increased clarity patterns especially where minor components lcerned. significant that starch gel runs involve a 1 hr period or overnight when urea is present, r electrophoresis and 3 hr for staining, whereas se acetate involves a total time of only about There is also a major difference in the amounttt of protein loaded, with about 5/zg being 3r cellulose acetate compared with 50/zg for used for starch gels. The cellulose acetate technique described above retains the simplicity of single baths for staining and dng which is a feature of the widely used destaini~ m S method. The separate fixing step and Ponceau le destaining baths of Fazekas et aL (1963) multiple have been found unnecessary. Coomassie blue, er, has the advantage :y (Fazekas (i however, g( of its sensitivity et al., 1963) which we estimate to3 be some fifty times greater than Ponceau S. he cellulose acetate We have successfully used the electrophoresis procedure at IpH values ranging
from 2-4 to 12 wit resolution of both and II). In contras membranes of ot Valentine, 1973), v Millipore membral handle wet or dry. in the degree of r( batches of membra the high-tyrosine pJ the storage globulil angustifoh [(Lupinus LltplnUS Electrc Electrophoresis method ccurrently tyrosine ]proteins c applicatk )lication and th of the Cc Coomassie [ proceedir to imp1 proceeding nique to (other keta
ut 8 M urea for the ~-tyrosine proteins (I nt report relating to cturers (Gilliam & d that the Beckman nly and are easy to ty has been observed ained with different ~roteins as diverse as ns from keratins and eed of the blue lupin acetate is the best examining the high;cause of its ease of tivity and simplicity procedure. Work is ication of this tech-
s R. G. & DAYTNERA. (1963)Two new s dures for quantitative estimati estimation of pl lectrophoretic strips. Biochim. 91. Biochim biophys, z 1-fOtglllb; rich I1~I1 in 11 glycine and J. 1¥1. M. ~lYl/,,ii) (1~ GILLESPIEd. rrosine from keratins. Comp. Biochem. PhysioL 41B, tyrosine 723-734. aatemary ammonium ]' I LLESPIEJ. M. ( 1 9 7 2 b ) The use of quaterna GILLESPIE in the fractionation ot of tyrosine-rich ethyl cellulose c( proteins )roteins from wool. J. Chromatogr. 72, 319-324. ! GILLIAMl~ Jnusual electroM. & VALENTINED. K. (1973) UI phoretk )horetic behaviour of proteins from honey hol bee hemolymph. Comp. Biochem. Physiol. 45B, 4( 463-466. reliability of molecuWEBERK. & OSBORNM. (1969) The reliabi lar wei~ght determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406--4412. E ST. GROq FAZEKAS DE
Key Word Index--Mammalian keratins ; high-tyrosine porcupine quill; proteins; mouse hair; guinea pig hair; p( merino wool; cellulose acetate; Coomassie brilliant blue.
