COMPARATIVE ANALYSIS OF SERUM PROTEINS BY CHEMICAL A N D ELECTROPHORETIC MEANS An Experiment in Biochemistry FABIAN LIONETTI and M. MOIRA DAVISON Boston University School of Medicine, Boston, Massachusetts
OCCASIONALLY an experiment is devised which not, only satisfies the requisites for proper instruction but also possesses elegance of design and simplicity of execution. These attributes plus enthusiastic reception by the student add up to unusual success. The rapid expansion of knowledge of the physical and chemical properties of blood proteins demands that the biochemical laboratory education include as many of the newer concepts as can be conveniently manipulated and assimilated by medical students with limited
were obtained in a majority of the 16 weeks of experimentation, and the experiment impressed the instructors as a most successful effort. For this experiment, the class was divided into groups of five or six students. I n outline, the experiment consisted of an orientation lecture on electrophoresis, demonstration of the instrument (Tiselius), interpretation of patterns, and presentation of photoprint copies on rectangular coordinate graph paper of the pattern for an experimental sample of pooled human serum. A graphic integration of the pattern curve was performed by each group, and the albumin-globulin ratio mas computed. A sample of the same serum was then analyzed chemically by each group, the A-G ratio was det,ermined, and results were compared with the electrophoretic value. Alpha, beta, and gamma fractions of the serum were similarly compared by the physical and the chemical method, although major emphasis was placed on the A-G ratio because of its diagno~t~ic significance in routine clinical chemistry. PROCEDURE
Preparation of Serum. Prior t o the class exercise, human serum was prepared as follows. A hospital clinical chemistry laboratory collected the sera remaining after required analyses were performed, until the volume of the pool reached about 200 ml. The blood was obtained from patients suffering from a wide variety of diseases, many of which caused no change in Poobd Human serum t,he total protein content and some of which caused a hypoproteinernia. The pool was filtered through a Ascending boundary. verooal butler, pH 8.6, r / 2 = 0.1 D6 Steriflow filter (Hormann) and divided into 10-ml. "Total protein" 490 Albumin 263 portions. Precautions were taken t o insure sterility of Total globulim 227 the samples, since it was desired to keep some a t least A/Q 263/227 = 1.16 four months. The serum was stored in 25-ml. sterile bottles at 1°C. Electrophoretic Analysis. The serum was diluted scientific training. Electrophoretic analysis of serum proteins by such students is hardly a practical problem 1 : 5 with veronal buffer a t DH 8.6. I'/2 = 0.1. and diat o be attempted in a given week. However, during lyzed against the same buffer for one week a t 7T.,the the past year we have aided our biochemistry class external buffer solution being changed a t appropriate in accomplishing not only electrophoretic analysis of a intervals. The dialyzed material was resolved for given sample of serum but also chemical fractionation 139 minutes in a 2-1131.cell, current = 6 ma. and potenand analysis as well. Moreover, unexpectedly good tial drop = 117 volts. A four-fold magnification of correlations between physical and chemical met,hods the Philpot-Svensson lens pat.tern was mimeographed =i
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on graph paper. By the method of weighing, the A-G ratio was estimated at 1.24; by the method of counting squares, a t 1.16. Electrophoresis Lecture. Each group of five students was given a one-hour informal lecture on the principles and uses of electrophoresis. The essential parts of the machine were described and a demonstration of the technique of filling the cell was given. The students were shown a wide variety of electrophoretic patterns, representing a monophasic system, multiphasic systems, normal and pathological human sera, and sera of various species. Graphs of pH versus mobility were shown to facilitate understanding of the isoelectric point. Each group was provided with a mimeographed pattern and directed to determine the A-G ratio by an optional graphical integration procedure and to correlate the value computed by this physical means with that obtained by chemical fractionation and anal-
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Chemical Detemination of the Serum Proteins. A detailed procedure for fractionating the serum proteins into total protein, albumin alpha globulin, gamma globulin, and beta globulin, and their determination with alkaline copper sulfate reagent (biuret) is given by Consolazio, et al.' The principle, apparatus required, reagents, procedure, sample calculations, and precautions are adequately described so that the student group, if provided with reagents, could individually accomplish the total procedure in four to six
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hnnr.
Comparative Analysis of Pooled H u m Serum by 3iu-t and Electrophoresis
Total protein
(In gmms/lW ml. plasma) Alhumzn Told &bulzn (T.protein alpha globulin - albumin)
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6.50 4.83 2.87 5.27 2.97 6.64 4.50 2.99 6.16 3.00 5.83 6.66 2.47 4.65 6.14 Eleetrophoretie analysis (methcd of eounling squares) mm.* 4.90 2.63 2.27 1.16
protein value minus the albumin, a high value for the latter resulted in an exaggerated high value for the A-G ratio. Nevertheless, when such pitfalls were pointed out to the students, the results in the hands of inexperienced workers were often gratifying. Popper, et ~ l .made , ~ a detailed comparison of the electrophoretic technique with chemical analysis b y the biuret method and found good correlations between the albumins, total globulins (and A-G ratio), the gamma glohulins, and the beta gamma (euglohulins) fractions. Poor correlations were found between alpha glohulins and beta glohulins. The 156 samples studied included 140 specimens from patients with liver disease and other disorders known to influence the serum proteins. It is possible to estimate the alpha and beta globulins from the measured quantities in the table, that is:
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The total protein value of the pooled human serum was 6.60 grams per cent, as determined by the microKjeldahl procedure followed by nesslerization (protein Serum alpha globuliu = (serum albumin + alpha globulin) - serum albumin conversion factor, 6.25). A standard curve was prepared by using bovine serum albumin in increments of (Serum beta globulin gamma globulin) = total protein - (albumin + alpha globuliu) 0.01 mg. from 0.76 to 7.00 mg. albumin per 3 ml. of sample analyzed. Using this curve, analysis of the Serum beta glob& = (serum beta + gamma globulin) - serum gamma globulin pooled serum measured 6.50 grams per cent by biuret technique. The curve was used weekly by each group However, errors in the measurements are too great t o after first verifying several points. permit accurate determinations of these quantities by Calculationand Correlation with Electrophoresis. The difference, and the student data were inadequate for chemical composition of the pooled serum sample de- this purpose. Nevertheless, the results derived were termined by five representative groups of five students most gratifying in that' the students were able to is given in the table. The composition computed by assimilate the rather formidable pedagogic concepts of the group from the electrophoretic pattern of the same electrophoresis, mobility, the isoelectric point, and serum is illustrated in the figure. The area under the chemical and physical fractionation of serum proteins curve was most conveniently obtained by counting in a surprisingly facile fashion. Much of this was accross-section squares. In a surprising number of in- complished by the expedients of actual manipulation stances, the more painstaking student obtained good and participation by the students. correlation of the physical and chemical methods. The most difficult part of the procedure was the deter- ACKNOWLEDGMENT mination of the albumin fraction, which required parThe authors wish to acknowledge the assistance of ticular care in avoiding high results, because of con- Dr. Matthew A. Derow of the Department of Microtamination of the fraction with precipitated proteins. biology. Since the total globulin was computed from the total POPPER, H., J. DE LA HUERGA, M. FRANKLIN, W. B. BEAN, ' C O N S ~ O ~C.~ IF., O ,R. E. JOHN~ON, AND E. MAREX,''MetaW. D. PAUL,J. I. ROUTH, AND F. SOHAFFNER, Am. J . Clin.
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bolic Methods," C. V. Mashy Co., St. Louis, 1951, pp. 123-6.
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Pathol., 20, 53C-8 (1950).
