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P R O T E I N RESEARCH s r u m s

Mhen I first went t o Kansas State College I was very much in te r - ested. I am not qui te sure w h a t t ha t means, but I do most of my work wi th poultry. I was interested when I first went t o Kansas S ta te College i n poultry meat, but a f t e r observing w h a t they do with poultry i n that section of the country, I decided tha t flavor, which is the thing that I wanted t o study, was not very important down there. t a s t e any chicken at a l l .

I am a poultry chemist there. That is my t i t l e on the books.

They barbecue a l l the i r chickens and they don't rea l ly

In addition t o that , the Home Economics Department was running a series of studies on tas t ing. O f course, i f any of you have ever been on a t a s t ing panel you realize that there is no f lavor there but some stewed chicken and it rea l ly does not have too much flavor. We devised a group of diets in an attempt t o vary that flavor. t i c a l l y took the f lavor out of the chicken. It was more o r less of a puri- f i e d d i e t , I w i l l c a l l it. meat again by adding various additives t o it, grains, etc.. because we found out t ha t the easiest way to add f lavor t o the meat of chicken was t o add about two shovel. fi l ls of chicken droppings t o each hag of feed and you ended up with a f a i r l y good tas t ing chicken.

We also devised a diet which prac-

Then we t r ied t o put the f lavor back in to the We dropped that

However, w e have been working on technics t o study protein, and we have been concentrating on electrophoretic technics. in te rs ted i n phosphorus, i n t rying t o f ind ways and means of determining where i n protein mixtures you would f ind these proteins. ta lking of' soluble proteins. the electrophoretic technic.

We were par t icular ly

We are, of course, You cannot do it w i t h insoluble proteins wi th

We had two things we could do. We could fract ionate proteins. If you have ever looked in to the l i t e r a t u r e and you have seen the various long- winded methods they have for f ract ionat ing proteins, you rea l ize tha t t h i s is a s o r t of discouraging proposition, and I did not want t o spend the next 20 o r 25 years doing so. So, taking the eas ies t way out, you might c a l l it the lazy man's way, we sat down and did a l i t t l e thinking about it, and we worked out a combination of electrophoretic technics with radio-chemical technics that I think some of you may be interested in .

I talked about t h i s w i t h D r . Hall before we came here, and he suggested that, since th i s would be quite a heterogeneous group, I take a few minutes i n the beginning and explain what I mean by th i s electrophoretic technic and how we apply radio-isotopes t o it, before I show you a piece of t he equipment and some of the typ ica l resu l t s t h a t w e have.

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(Slide) Don't let t h i s diagram scare you. This is j u s t a diagram- matic sketch tha t I should l i k e t o use t o indicate t o you something tha t i s s o r t of second nature t o me, but I f ind that it is not second nature t o most other people. What I have is j u s t an U-tube here, and i n t h i s U-tube I have a solution. The cross-hatch part is a mixture of two materials which we c a l l proteins A and B. there is actually nothing in between here. This is j u s t where the two solu- tions come together t o form w h a t we c a l l an interphase.

graph. solution and you at tach an electrode so that you have a minus charge o r voltage on one side and, say, a posit ive voltage on the other s ide and you apply a potent ia l across it. If these materials o r proteins have a charge on them, they go t o the posit ive electrode i f they are negative and t o the negative electrode if they are posit ive. They move away f r o m each other. The f ront end and the back end of t h i s slug of protein material w i l l move ju s t as f a s t as each other. In other words, as A moves up f r o m t h i s posit ion t o th i s position, it will move down on the other side. distance, f o r instance and it w i l l move tha t short distance down on the other s ide. trophoresis

The material above that is j u s t a buffer solution, and

When a light shines through this solution t h i s shows up i n a photo- Suppose you take t h i s I w i l l show you w h a t t ha t looks like l a t e r on.

B moves only a short

This is w h a t they c a l l an ascending and descending boundary i n elec-

(Slide) Now I w i l l straighten the tube out, so I can show you w h a t happens as far a6 the concentration is concerned. YOU see there is protein here and there is protein here and a l l t h i s represents is zero protein, and then when I get t o th i s interphase I go from zero protein t o whatever concen- t r a t i o n I have i n there. This is measured by an index of refraction. It does not show up on the photographic plate. This l i t t l e peak here.

It shows up l ike this.

(Slide) This i s w h a t happens when we have two i n there t h a t move You see th i s first one is the most concentrated and at different speeds.

you get this f i r s t peak. of t h e two kinds, the two peaks. photographic plate.

Suppose I not only have protein materials i n there but I have t h e m marked. Suppose I have been feeding an animal, let 's say, radio- ac t ive phosphorus. be act ive and some of them may not be active. Here I have my electrophoresis s e t up and the materials are moving i n th i s direction. centrations moving down the l ine , as I did before, except t h a t I have one more. two are radioactive and the first one is not, if I d r i l l a hole i n t h i s c e l l and I put a counter r igh t at t h i s point I can count them as they go by. In other words, it i s ju s t as if you had 50 men running and t en of them could run 10 miles an hour, 10 of them could run 20, and 10 of them could run 15. They would arrange themselves in to groups eventually. t he first group t h a t went by was not radioactive, but t he next two w e r e and it would show up as the t o t a l number of counts on a Geiger counter.

Then the other one which is the l e a s t concentrated It shows up as two peaks l ike t h i s on the

(Sl ide)

These proteins ai= now active. A t least some of them w i l l

Here I have the con-

Here i s the way it w i l l show up on a photograph. Now if only the last

It so happens that

(Slide) This is a picture of an end view of an electrophoresis ce l l , and th i s gives you an idea as t o w h a t these peaks look like i n a

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photograph. This is the hole i n the side of the ce l l . than .1 of a millimeter thick r ight there. that we can spot in the ce l l , you can put a Geiger counter alongside and count them. Then you can t e l l which of these peaks have the phosphorus and which do not have the phosphorus o r the sulf'ur, whatever you want t o measure, depend- ing on whether they a re radioactive o r not.

They start r ight down here, and t h i s is the tube that I mentioned. This is pyrex glass. It is l e s s

As these various peaks mve by

I w i l l show you the setup of the c e l l itself I think i n the next 6 l ide .

(Slide) This is a glass c e l l . It is rather d i f f i c u l t on the photo- graph, but you see there the ground section of the ce l l . portion r ight here where the solution moves by the hole, and t h i s i s the gadget on the side, the plexiglass frame t h a t holds the Geiger counter.

Here is the center

(Slide) These s l i des are not very w e l l adapted t o t h i s room. C a n you see t h i s back there? This is the long tube t h a t contains the Geiger counter and that f i ts r ight in next t o the c e l l . This goes over t o these electrodes on the side.

I am having d i f f icu l ty , being close t o it.

This i s the U-tube that I mentioned previously.

(Slide) This is good t o measure phosphorus but it would not be much good f o r su l fur and material8 that have a low count, So what we d id i n t h i s case was grind a hole i n the side of the c e l l . In t h i s case you f ind a fr iend who i s a dent i s t and you go i n and use his dental too ls and you grind a hole r ight i n to the side of the c e l l and cover it with j u s t a l i t t l e piece of polished styrene, they c a l l it, in t h i s thing r ight up close t o the side of the hole.

This is a frame which holds an end window counter, as

(Slide) You see t h i s is the same U-tube except now instead of having the Geiger counter tube f la t againat it, we have a4 end window counter.

(Slide) This is the way t h i s one looks i n the apparatus. The glass- I want you t o

The only way you can photograph glass is t o have it di r ty , I ware i n t h i s par t icu lar picture i s out because it is clean. notice tha t . found out. the window counter f i t t ed t o the apparatus . This was taken when it was very clean. So there it is again with

(Slide) Those are the various peaks, for instance, of a solution of various protein components moving past t h i s par t icu lar hole i n the tube. Anyway, as the peaks move by th i s hole you take the nuxiber of counts at each point, f o r instance, A, B, C, D, e t c .

As I mentioned, t h i s is an electrophoretic picture.

You will notice tha t this picture has A, B, C , D, e tc . along the top. For instance, A gives us what we c a l l the background count which f o r a l l prac t ica l purposes is zero or if it i s n ' t zero w e subtract first fraction. and the second f ract ion. t r ac t ing one f rac t ion f r o m t he t o t a l number of f ract ions you can f i n d the per- centage of phosphorus i n t h i s par t icu lar mixture.

Every place that is marked we measure.

it. B gives us the amount of radioact ivi ty i n t h i s C gives us the amount of radioactivity i n the f i r s t f rac t ion

You see, by going through t h i s procedure and sub-

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You can also get the amount of protein materials supposedly present I th ink I have a picture by measuring the area under these various fractions.

coming up where we did that axid 1 . w i l l show you how it works out,

(SUde)

There 13 not much phosphorus here.

No, t h i s is mother one. I think t h i s is egg white, al- thoueh I am wt certain. You can see that the background i n this par t icu lar case was 30. There is not much phosphorus here. 38 he=. So we have four f ract ions i n here which do not contain phosphorus. We begin t o move over t h i s back frac- t i o n here after the analysis has progressed, and then we see tha t our phos- phorus has peaked, so we can pinpoint where the phosphorus is i n t h i s material.

Here is an analysis.

As you go down, this is 31.

(Slide) This j u s t happens t o be blood serum i n th i s par t icu lar instance. This is f rac t ion 1, 2, 3, 4, 5 and 6 . This is ac tua l ly the amount of phosphorus we can determine is i n each of those frac- t ions

(Slide) phorus and running a nitrugen determination t o get the nitrogen-phosphorus r a t i o i n these proteins , Thfs, for instance, is the amount of nitrogen i n each of those components 1, 2, 3, 4, 5 and 6 . bears no relationship t o the amount of phosphorus you have present there. So in t h i s manner, you see, we were attempting t o determine which of these f rac- t ions, whether it was the large o r the small, contains the largest amount of

It gives you another example

You eee that it is very possible by determining the phos-

You w i l l notice down here it

PhOSphOrUS

(Slide) This is Just another one. w i t h another picture.

(Slide) This is the part that interested me. Dr. Deatherage was ta lking t h i s morning about h i s calcium, magnesium, e tc . It was possible f o r us t o s h i f t t h i s phosphorus, these various components, one way o r another t o determine which of these components, f o r instance, calcium would react with by determining the amount of phosphorus i n these various fract ions with and without calcium i n t h i s pa r t i cu la r buffer mixture of proteins, and it struck me that t h i s m i g h t be one way that h i s group could approach t h i s problem of which one of these f rac t ions i n the mat, the calcium and magnesium were attached to .

These black bars w i l l give you the a m w t of phosphorus in each of these fractions, A, B, C, D, etc., when calcium is not i n the solution. These small ones give us the amount of phosphorus i n those par t icu lar f rac t ions up there when calcium was added t o the solution. You w i l l notice that it stays low u n t i l you go right t o there, and ac tua l ly you move a f rac t ion t h i s way and by so doing you can determine which one of those components t h e calcium is attached t o ,

I j u s t happened t o think, when I heard him ta lk th i s morning, t h a t by using the Juices from the cooked meat and t h e raw meat o r the cured meat, etc. , you might be able t o f ind 801118 r a t io here a8 t o which of these fract ions the calcium was attached to .

This is a rather expensive wgty of doing business real ly . We de- cided tha t we would have t o have an easier way i f we were going t o get anywhere.

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So we went i n t o s ta rch gels. trophoresis. mixed with water and allowed t o settle and it comes out as a sor t of paste. This is not that. can cook it. qual i ty eelat in .

Probably most of you have seen starch elec- By that I mean the granular starch.

You nul it in to a trough and it turns out Just like a good

Starch t h a t has just been

This i s a gel, a hydrolimed starch. You can wash it. You

It is nice and c lea r and bubble-free.

(Slide) You have a trough like t h i s up here. This looks down on t h i s trough. l i t t l e chunk out with a knif'e and it lifts out j u s t as easy as can be, j u s t as if you were s l i c ing gelatin. run this in the same m e r that you ran the mre complicated type of elec- trophoresis. and you can analyze it f o r radioactivity, you can analyze it fo r nitrogen -- f o r almost anything you want. You can even recover your sample if you so de- sire by taking these samples and freezing them and pouring them o r chopping them up in to smaJ.1 pieces and mixing them wi th a l i t t l e buffer and then l e t t i n g them soak fo r a w h i l e .

You f i l l t h i s trough with c lear ge la t in solution and you cut a

You pour your sample into that. Then you

When you have finished you dumg it out and slice it into pieces,

It works p re t ty well. I have some of t h e m here.

I thought it would be better if I jus t showed you the resu l t s rather than trying t o make a slice from it. A couple of t h e m have radioactivity i n them, so don't open them. I was able t o get them away from my graduate students only by t e l l i n g them I would bring t h e m back.

I should l i ke t o have them back, if I may, because

3% you will t i p them up you will notice that I have an electro- phoresis here that has been run on these s tarch gels.

Then I have developed it w i t h a Medo black which is a protein dye. You can get any number of f ract ions i n t h i s material. i en t way of doing it. You can set yourself up i n business with t h i s particu- lar type of electrophoresis f o r a couple of hundred dol lars w h e r e a s the other type costs many thousande, but this is very easy t o do.

It is a very conven-

I w i l l be down a t the other end and col lect them, I don't want them t o get l o s t .

Well, I hope I have been able t o show you a l i t t l e about a technic that many people seem t o be af ra id of. Just run away f r o m you and they refuse t o l i s t e n and t r y t o understand w h a t yau are t rying t o explain. a captive audience and I don't have t o worry about i t s running too far.

The minute you mention it they a l l

I am B l i t t l e lucky here t h i s afternoon. I have

I)€?* SCHWEIGEilU!: We have, i n opening the discussion, two short reports from two other groups, who will describe b r i e f ly some of t h e i r thinking wi th respect t o research approaches with meat proteins. t h i s off w i l l be our good friend, Joe Kastelic, who w i l l share with us some of his thinking in this regard in a very short report.

Leading

Joe.

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DR. J. KASTEW;C: Ladies and Gentlemen: Frankly, I dontt know just why I am here. represents very l itt le the kind of experience that some people have de- scribed here in telling about the work they have done on meats.

I haven't mch to say and what I have to say

From this discussion I have deliberately left out many aspects of research approaches with meat proteins, since fronthe program I aElsumed that some of the thing8 that one could talk about would be dis- cussed by other speakers