Idea Transcript
GRAVIMETRIC METHODS FOR THE DETERMINATION OF TOTAL BODY PROTEIN AND ORGAN PROTEIN* BY
(From
T. ADDIS,
L. J. POO,
the Department
W. LEW,
of Medicine, Stanford San Francisco) for publication,
D. W. WEN
University
December
Medical
School,
26, 1935)
Although much has been learned about protein metabolism by such indirect methods as Nz balance experiments, there are still elementary questions to which no answer can be given until direct measurements of the protein content of the various organs and tissues of the body have been made. With the exception of methods for protein in plasma (1) and muscle (2) the gage of the protein content of the body has hitherto been taken as given by its nitrogen content. But even if all non-protein nitrogen could be removed, it is evident that this is not a satisfactory procedure for estimating the sum of a mixture of proteins whose nitrogen concentration is known to vary from 10 per cent to 18 per cent. On this account and in order to investigate certain problems which require a direct approach and precise results the following gravimetric methods have been devised. They have been gradually developed during the past 5 years and in their present form have been shown to be reliable and accurate. The general procedure is first to prevent autolysis and to render the proteins insoluble by immediate immersion of the carcass and excised organs in a boiling 0.5 M sodium acetate-acetic acid buffer solution kept at pH 5. The material is then reduced to a form in which it can be sampled by methods which vary with the physical character of each organ or part. Water-soluble substances are washed out with acetate buffer, fats and acetate with hot alcohol, and finally water is removed by drying in vacua. The remainder is weighed. Non-protein material which cannot be removed, such as minerals derived from bone, and glycogen * This
work
was
aided
by a grant 497
from
the Rockefeller
Foundation.
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(Received
AND
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that cannot completely be washed out from the liver are determined and subtracted. The material thus measured is regarded as giving the nearest approximation to the amount of the protein structure required for the manifestation of vital phenomena, the essential machinery stripped of storage material and accessories and separated from the medium within which it operates. Sampling of the material can be avoided whenever the mass is not too great. In our work we were anxious to eliminate, as far as possible, the factor of individual variation, so we have used a highly standardized strain of albino rat and have made each measurement on the mixed organs and carcasses of thirty animals. They were males between the ages of 90 and 110 days and under ordinary conditions their carcasses weighed about 5000 gm., their livers 250 gm., and their kidneys about 45 gm. These quantities were all too large to be conveniently handled without sampling. In the case of the hearts, however, which did not weigh much more than 20 gm., and of course in all smaller organs it was possible to deal with the whole material without sampling. Liver Proteins-lt is not necessary to reduce the livers to a fine powder before the material is sampled, because in this case complete homogeneity can be attained by converting them into a form which corresponds to a p&b de joies gras. Each boiled liver is ground in a mortar with enough buffer solution to make a thick soup and all of them are transferred to the bowl of a household utensil known as a “Kitchen Aid.” This is a motor-driven apparatus which keeps a fenestrate paddle revolving through the soup. As the stirred brei thickens it becomes a stiff paste and finally attains a putty-like consistency. In that state it gathers together into a self-adherent ball which can be transferred without any loss in handling. The whole mass is then quickly divided into parts of convenient size, placed in air-tight stoppered vessels, and weighed on an analytical balance. The samples taken varied from 2 to 6 gm. in weight, corresponding to from 2.5 to 7.5 gm. of fresh liver. They were transferred from the weighing bottles to 50 cc. centrifuge tubes, and ground in about 35 cc. of 0.5 M sodium acetate solution of pH 5 by means of a stout rod. There was nothing left now of any cellular structure. Under the microscope only a suspension of particles could be seen. This suspension was left overnight at 0”
Addis, Poo, Lew, and Yuen
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and was centrifuged the next morning. The opalescent supernatant fluid on which some fat floated was decanted through a Whatman seamless fat extraction thimble. The process of grinding in buffer solution, centrifuging, and decanting was repeated four times. The liver paste was then washed with 95 per cent alcohol from the centrifuge tube into the thimble which was plugged with cotton-wool. This thimble and the plug of cottonwool had previously been extracted with hot alcohol, dried, and After being washed with absolute weighed in a weighing bottle. alcohol, the material in the thimble covered by the cotton-wool was extracted in a Soxhlet apparatus for 18 hours with absolute alcohol. No measurable amount of material was removed by The thimble longer extraction or by the use of other fat solvents. containing the protein was then returned to its weighing bottle and the alcohol removed by evaporation in an oven at 110”. There still remained the difficulty of drying protein to a constant weight. This was only overcome after we had attached a watercooled oil pump to a vacuum oven and were able to maintain an almost complete vacuum at a temperature of 80” for 48 hours. Under these conditions further drying has no measurable effect and the protein may be weighed. In the case of the liver we did not succeed in completely washing out all glycogen. Even when the grinding and suspension in buffer solution were repeated eight times the supernatant fluid still contained a little glycogen, and so it had to be measured and subtracted from the protein-glycogen weight. The dry protein takes up water with great rapidity and the transfer of part of it to another weighing bottle must be done as quickly as possible and either in a box filled with dry air or within an oven. Quantities of from 0.5 to 1 gm. were taken for analysis. The method of Sahyun was used (3). In fasting experiments and in a series in which thyroxine was given no measurable quantity of glycogen was found in the washed protein. In the This liver protein method has a high degree of accuracy. livers from each group of thirty rats eight protein determinations were always made and the mean deviation from the average was 0.3 per cent. A similar order of accuracy was reached for the protein in other organs. Kidney Proteins-The capsules of the kidneys were removed and
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* When the brain and testicles are treated separately, a small amount of protein passes through the thimble with the acetate buffer. This protein is retained on refiltering after the filtrate has been heated with 3 times its volume of alcohol.
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they were opened and blotted with filter paper before they were weighed and coagulated. The sampling is most conveniently accomplished by grinding the boiled kidneys with a little buffer solution in a large mortar until the material adheres together in a putty-like mass which can be subdivided, sampled, washed, extracted, and dried as in t.he case of the liver. Only traces of glycogen too small to measure were found in the dried protein and as no variation under widely different dietary conditions was observed, we have not found it necessary to carry out glycogen determinations as a routine procedure. Heart Proteins-The heart was cut out always by the same individual, using the same technique. The apex was held by forceps and the heart pulled up out of the pericardial sac, while the inferior vena cava and pulmonary vessels were divided by scissors. The aorta and superior vena cava were cut just above the still beating auricle. Both ventricles and auricles were opened and any adherent blood removed with cotton-wool before the heart was weighed and boiled. Then all the thirty hearts were ground together in a mortar and separated into four parts which were weighed and transferred to four centrifuge tubes for washing. The total heart protein was then determined in the same way as for the liver and kidney. Proteins of Gastrointestinal Tract and Other Organs’-In most of our work the spleen, adrenals, pancreas, bladder, seminal vesicles, and testicles along with the abdominal and pelvic fat pads were removed en mass with the gastrointestinal tract and all these organs and tissues were boiled together in buffer solution. The whole tract was then pulled out into a straight line, opened with a razor blade, and the contents washed out under running water from a tap. After drying overnight on glass plates in a current of air at a temperature of 64”, the whole mass was collected in the bowl of the Kitchen Aid and stirred with acetone, transferred to a Buchner funnel, the acetone filtered off, the material returned to the Kitchen Aid, stirred with ether, and the
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ether filtered off. After this treatment it was found that everything could be reduced to a fine gray powder by grinding in a mortar. The whole powder was allowed to become air-dry, weighed, and the samples treated in the same way as in the case of the liver. Blood Proteins-All the blood which could be obtained from the opened abdominal aorta while the heart was still beating was collected. It was centrifuged at high speed and the serum drawn off and weighed. Acetate buffer (5 times the volume of serum) was added and the protein was coagulated by raising the temperature to the boiling point, while the mixture was being stirred. The precipitate was filtered off on a Buchner funnel and washed with hot absolute alcohol. The material then contracted into a sponge-like mass which could be sampled without loss. The method thereafter was the same as that used for the liver, kidney, and heart. The clot was weighed and boiled in buffer solution which was later filtered off under pressure. It was then washed with acetone, transferred to a mortar, ground to a powder, allowed to become air-dry, weighed, sampled, and treated as were the organs. Carcass Proteins-The carcass is the exsanguinated rat with the heart excised, stripped of all abdominal and pelvic organs, and of the removable abdominal and pelvic fat. Each of the thirty carcasses was boiled in buffer solution and then chilled by storage in a room at -2’. While still cold they are minced in a meat chopper. If they are ground while warm some of the “gelatin” may be lost. The mince is spread on glass plates and dried at 64” in a current of air for 24 hours, and is thus reduced to about 40 per cent of its original weight. The material is then stirred with about 3 times its weight of acetone. The acetone is filtered The off and the remainder is stirred with ether and filtered again. material is now ready for grinding to a fine powder in a Wiley mill. The ether must be removed by suction. If it is too dry there may be a slight loss by the escape of a very fine powder during the powdering process and it may then be necessary to sprinkle it with a little acetone. In the mill the powder is passed through a fine mesh screen. It ordinarily weighs about 1500 gm. It is collected in an open can and when it has become air-dry it is weighed on a Sartorious balance sensitive to 0.1 gm. It is not convenient to
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take samples of more than a few gm. in weight and it is evident that in material consisting of such diverse constituents as hair, bone, connective tissue, and muscle one would not be surprised to find considerable errors in sampling. At first we attempted to attain thorough mixing by shaking the powder. The average discrepancy in the first eleven experiments between determinations on the same materials was 0.7 per cent. But it was later observed, when we were dealing with very emaciated rats in which the fur was a relatively greater part of the whole, that this shaking led to a separation and balling together of the hair particles, and in these experiments the average error in one case rose to as high as 2.9 per cent. Since then shaking has been given up and the powder from the mill has been layered over a large area after being passed through a fine screen. Even so the sampling is not very accurate and we have felt it best to wash a minimum of eight samples from each lot of carcasses. The samples are placed in centrifuge tubes, suspended in buffer solution, and washed in the same way as are the organ proteins. The supernatant fluid contains some hair particles but these are not lost since the fluid is decanted through a fat extraction thimble. After Soxhlet extraction with hot alcohol, to remove the remaining traces of fat, and drying in vacua at SO” the protein and the bone minerals are weighed. The extraction with the acetate buffer solution at pH 5 had liberated COZ from the calcium carbonate of the bone, but it is not necessary to try to define the exact nature of the mineral residue, for the total weight can be determined by dry ashing. For this purpose the thimble containing the protein and minerals is placed in a double porcelain crucible as recommended by Stolte (4) and heated over a flame until the major part of the carbon has been oxidized. It is then transferred to a cold muffle furnace and the temperature raised over the next 3 hours to about 770” before it is cooled and weighed. The weight of minerals subtracted from the proteinmineral weight of the sample gives the protein content. Up to the time when this method was devised the protein content of the carcass was determined indirectly from the Nz content of the washed and alcohol-extracted samples. The preparation of the carcass powder for sampling is a someIt is none the less what tedious and time-consuming procedure. necessary if measurements of the distribution of protein in the
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TABLE Protein
Distribution
in Thirty
I
Rats
Reared
on Adequate Protein weight rat
Carcass ........................... Liver ............................. Kidney. .......................... Heart ............................. Remainder ........................ Total...........................
/
per
gm. 194.70 8.98 I .48 0.83 44.22
gm. 35.82 1.79 0.26 0.14 3.72
250.21
41.73
Diet Protein distrimtion, gm. per 100 gm. total protein
85.9 4.3 0.6 0.3 8.9 100
body is an essential instrument for a direct attack on many important problems in protein physiology. The nitrogen concentration in protein preparations under varying experimental conditions averages 16.02 per cent for the liver protein, 15.91 per cent for kidney protein, 16.20 per cent for heart protein, and 16.60 per cent for carcass protein. We had hoped that we might be able to wash out most of the minerals after boiling the organs at a pH of 5 in acetate buffer, but the ash content was only reduced to about 30 per cent of the amount present in the unwashed liver, to 23 per cent in the kidney, and to 18 per cent of the original amount in the case of the heart. The ash content of the dry protein preparations was accordingly high, 2.2 per cent for liver, 2.0 per cent for kidney, and 1.3 per cent for heart protein.
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body under widely varying conditions are to be compared. It would for instance be of no avail to obtain an accurate measure of the protein in the liver in well fed rats as compared with the protein content of the liver of fasted rats if we compared these quantities in terms of, say, 100 gm. of body weight. Casual inspection is sufficient to show that 100 gm. of the well fed animals containing a relatively large proportion of fat is far from being the equivalent of 100 gm. of the lean fasted rats. A valid comparison requires that these organ quantities be expressed as gm. per 100 gm. of the total body protein of the individuals from which the organs were derived, and, of course, apart from any consideration of organs or parts, the determination of the protein of the whole
504
Gravimetric
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Table I gives the protein distribution in a group of thirty between the ages of 90 and 110 days, reared on an adequate
rats, diet.
SUMMARY
1. Gravimetric methods for the measurement of the total protein of the body or of any of the organs of the body are described. 2. The total protein content and the protein content of various organs of well fed rats are given.
1. Barnett, C. W., Jones, R. B., and Cohn, R. B., J. Exp. (1932). 2. Janney, N. W., J. Biol. Chem., 26, 177 (1916). 3. Sahyun, M., J. Bid. Chem., 93,227 (1931). 4. Stolte, K., Biochem. Z., 36, 104 (1911).
Med.,
66, 683
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BIBLIOGRAPHY
GRAVIMETRIC METHODS FOR THE DETERMINATION OF TOTAL BODY PROTEIN AND ORGAN PROTEIN T. Addis, L. J. Poo, W. Lew and D. W. Yuen J. Biol. Chem. 1936, 113:497-504.
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