t)y analyses of standard protein solutions and standard control sera. Table I1 lists the means and standard deviations of various protein concentrations estimated by both procedures based on 24 determinations. The agreement for the concentrations of albumin, total globulins, and total proteins is very satisfactory. The electrophoretic method yielded a higher degree of precision than the chemical fractionation procedure. N O R M A L RANGES
Serum was collected from 40 presumably healthy persons, 20 of each sex, in order to detcrmine the normal i'anges of electrophoretic protein fractions. ,4n attempt ivas made to base the selection on the agc distribution of the general population; the average age was 35, S o two persons came from one family. Table I11 summarizes the data ohtained. The ranges were calculated to include 95aJ, of the persons chosen. The albumin-total globulin ratio, frequently used as a standard for protein halance, yielded a mean i. 2 standard (leviation of 1.34 f 0.19. Comparison of '1'ahl(~s I1 and 111
revCali: that the standard deviations of the normal ranges vary only u p to three times those of the precision of the method. This is difficult to reconcile with the finding of Strickland et al. (9) t h a t corresponding protein fractions from different sera varied widely in their abilities to bind the same dyes. They concluded t h a t large errors can result from dependence on this method which can be expected to detect only gross abnormalities in serum protein distribution. Lissaminc Green was not one of the dyes studied. Ehrmantraut ( 3 ) has calculated tht. mean normal values of serum protein fractions, expressed as percentages of thc total, obtained from the normal values from 36 published studies on paper electrophoresis. When compared with those det,ermined in the present investigation, the agreement is good (Table 111),the largest difference being in the a2-glohulin fraction. Histograms werc conptructed to determine the frequency distriliution of the normal values of each fraction; three appeared to be normal and two were skewcd. Since electrophoretic fractions coristitute the 511111~of many subfractions. thcir signi-
ficancr 1s difficult to ai.es+.. X o significant differences between sexes were found, but after completing over 200 analyses covering all age groups, a tendency has been noted for the albumin to fall and both a2-globulin and yglobulin to rise with increasing age. LITERATURE CITED
(1) Aron~son,T., Gronwall, -4., Scand. J Lab. (Tin. Znuest. 9, 338 (1957). ( 2 ) Brackenridee. C. J.. h 4 1 , . C:HEM. 32,
-
1353 (1960). (3) Ehrmantraut, Ei. C., "The Clinical Significance of Paper Electrophoresis," ~
Fipinco Division, Beckman Instruments, Inc.. Palo Alto. Calif., 1958. . .4cta 3 * 450 , , (1958).
( 5 ) Laurell, C. B., Laurrll, S.,Skoog, S . , Clin. Chern. 2 , 99 (1956). (6) Milnr, J., J . Bid. Chem. 169, 5% ( 1947). ( i )Owen, J. A,, Analyst 81, 36 (1956). (8) Rapp, K. I]., Memminger, 11. AI., A n i . J . Clin. Path. 31, 400 (19,59). (9) Strickland, R. I)., Podleskl, T. H.,
Gurule, F. T., Freeman, 31. L., Childs, JV. A , , ASAL. CHEJI.31, 1108 (1950). (10) JVolfson, IT7, Q.) Cohn, C., Calvary, I,:., Ichiba. F., .1 T U . J . ('!in. Path. 18,
Variable Dye Uptake in the Quantitative Analysis of Abnormal GIo b uI ins by Cel I uI ose Acetate EJectrophoresis I
COLIN J. BRACKENRIDGE Biochemistry Department, Royal Perth Hospital, Perth, Western Australia
b Examination of 28 serum samples containing abnormal globulins has shown that paraprotein dye uptake is variable owing to instabiliiy. Since prediction of dye-binding behavior seems impossible, the total protein content should b e determined by an independent method to arrive a t the concentration of the abnormal component.
I
THE previoudy described procedure for the quantitative estimation of human serum protein fractions if ), it has been assumc3d that no irregulaiitg in dye uptake occurs other than small contributions b y differing amounts of individual proteins within 3. given electrophoretic fraction. The observation t h a t the standard deviations of the normal physiological ranges of any of the fractions never exceeded t h t w times thow of thr. pi~cision of
x
thv method supports this claim. ?'her,r is, h o w v e r , the possibility that on rare occasions in normal persons, or in disease states, conditions might arise to invalidat'e the assumption. Physicochemical instability, molecular aggregation, or protein-protein interaction cannot be discounted from sometimes occurring in vitro or as a methodologicaal artifact. The present investigation deals with the dye-binding properties of the socalled serum paraproteins which are abiiornial in a quantitative or qualitative sense depending on whether they are formed by the selective increase of a particular protein component or as a different species from those present in normal and most pathological sera. '1'0 this class belong the cryoglobulins, macroglobulins, myeloma proteins, and aa-glohulins ( 5 ) . The latter are unstable prot'eins 1%-hichmigrate b e t w e n the a?- and 8-globulins! while t h r othcre
have mobilities of a?-,6-, or y-globulins or intermediate fractions such as AI-type paraproteins which migrat'c between 6and y-globulins. Although sevwal quantitative electrophoretic studies of such proteins have been published (a, 3 ) , scant attention has been paid to their d p b i n d i n g capacities. The work to be drscribcd arose from the question of whether their dye uptake was regular 01' variable, and, if the former, to which fraction they bore the most similarity. PROCEDURE
Sera of patients suffering from essential cryoglobulinemia, maci oglobulinemia, and myeloma are subjected to the quantitative electrophoretic procedures already described ( 1 ) and the protcin pattern is inspected. I n most cases a discrete abnormal band is visible in t h r g l o h l i n region. If it is superimposed on one of the fractions, the segments arc out out and thc dye is eluted as usual. VOL. 32, NO. 10, SEPTEMBER 1960
1359
This will include the normal fraction, but often i t is absent -as in 7-myeloma paraproteins-or present in small amount compared with the abnormal component. If the paraprotein has a mobility intermediate between two globulins, i t is cut out and eluted like the normal fractions. If no discrete band is apparent, the five fractions are rstimated as usual. hleanwhile the total protein concentration is determined on the same samplc of serum. A method such as the biuret copper sulfate is perhaps preferable to the Kjeldahl nitrogen estimation because of the uncertainty of the protein-nitrogen content, although the former method is a measure of peptide nitrogen and not of the basic amino nitrogen involved in dye-binding. The weight of Lissamine Green taken up by the abnormal band is then related to the difference between the total protein content and the sum of the other levels obtained by electrophoresis. If no abnormal component is visible, the dye uptake of each globulin is compared with the total protein content less the sum of the other protein concentrations. When this relation is characteristic of a normal globulin, the abnormal component is either present in negligible amount or has the same dye uptake as the fraction in question. RESULTS AND DISCUSSION
Figure 1 summarizes thc results obtained on 28 paraproteins. The data appear to be classifiable into four distinct groups, which are numbered in order of decreasing dye-binding capacity. Class I comprises 12 individual proteins which have the same dye-binding capacity as normal serum y-globulin. Class I1 consists of three proteins which have a somewhat similar dye uptake to p-globulins. Class I11 contains 11 proteins and class I V only two. Table I lists the distribution of the various types of paraproteins within these classes and according to their mobilities. Classes I1 and IV consist of 7macroglobulins and ,%myeloma proteins, respectively. Apart from this, the three types are evenly distributed between classes I and 111, and there is 110 obvious correlation between class, type, and mobility. Less than one quarter of the total
550
4 50 50
i
' *
I
I
I
I
I
I
I
II
I
1
N1 0 N lo 4 C O N CPROTEi ENTRAT
111
I'
I
I
l2
l3
Figure 1. Uptake of Lissamine Green as a function of serum paraprotein concentration in grams per 100 ml. Roman numerals refer to dye-binding class
number of pioteins studied migrated as diffuse bands; with one exception, all of these had a concentration of less than 2 grams per 100 ml. and belonged to class I. With reference to those paraproteins superimposed on normal fractions-usually y-globulins, it is possible that some of the niembeis of class I11 consist of a mixture of y-globulins and class IV proteins. This should not be a common occurrence since the normal fraction is seldom elevated in the presence of closely adjacent abnormal components; it is particularly unlikely t o OCCUI' a t levels above 2 grams per 100 ml. The four apparently regular groups of dye-binding paraproteins suggest that they are not completely heterogeneous with respect to each other: pending further investigation it is considered that some classes represent vai ious states of aggregation. This is supported b y evidence of instability. One myeloma protein F a s found to belong to class I on first investigation. The serum was then frozen a t -20' C. overnight following which i t behaved as a class I11 protein. Subsequent incubation at 37' C. for several hours gave rise t o a class I1 behavior. During these transitions the paraprotein concentra-
tion did not alter perceptibly wheieas the dye uptake varied by as much as 50%. Pedersen (4) has also noted the variability of aggregation of a myeloma protein under the influence of physical conditions. I n view of these effects, and since it appears impossible to predict the dyebinding characteristics of parapi oteins, i t is suggested that the total protein concentration of a dysproteinemic serum should he determined by a n independent mcthod and compared with the electrophoretic result. Agreement does not preclude the presence of a class I paraprotein, hut a difference in excess of the sum of the experimental eriors provides evidence for the evistencc of a class IT to 1V globulin. ACKNOWLEDSMENT
The author greatly appreciates gifts of sera from I. Mackay, Walter and Eliza Hall Institute, lfelbourne; J. A. Owen, St. Vincent's Hospital, Melbourne; F. J. Radcliff, Royal Korth Shore Hospital, Sydney; and W. Roman, Institute of lledical and Veterinary Science, Adelaide. Special thanks are due to D. H. Curnow, Royal Perth Hospital, Perth, for valua1,le assistance given in connwtion with the manuscripts. LITERATURE CITED
Table I.
Distribution of 28 Paraproteins According to Class and Globulin MobiIity
I I1 111 IV
1360
Macroglobulin
Myeloma Globulin
ClasP B
hl '2
2 2
ANALYTICAL CHEMISTRY
5' 4
Cryo-
globulin
Y
5 3
4
Total 12
3
1
11 2
(1) Brackenridge, C. J., ANAL. CHEM. 32, 1353 (1960). ( 2 ) Conn, H. O., Klatskin, G., A n i . J . Med. 16, 822 (1954). (3) Griffiths, L. L Brews, V. .A. I,.. J. Clin. Pafh. 6 , l h (1953). (4) Pedersen, K. O., Cold Spring Hnrbor S y m . Quant. B i d . 14, 140 (1949). ( 5 ) Sunderman, F. W.,Jr., Sunderman, F. W., Ann. Int. -Wed. 51,488 (1959). RECEIVEDfor review March 2.5, 1960. .iccepted June 23, 1960.
