8
A LABORATORY EXPERIMENT ON EXCHANGE AND BINDING OF Ca" BY SERUM PROTEINS R. E. HEIN and R. E. CLEGG Kansas State College, Manhattan, Kansas
Tm
experiment described in this paper has been in use for five years by classes in a course entitled radioactive tracer techniques. While the techniques were worked out because of research needs and have been described in part in other publications (1-S), the experiment is of considerable value to tracer technologists because i t
NYLON SQUARE Tim* I.
The Position of the Didyzinp Sack Prior to tha Foldine of the Nylon Squva
demonstrates the following points: (1) the rapid exchange of ionic calcium with serum protein fractions; (2) a rapid radiometric method to determine per cent ionic calcium in serum fractions; and (3) the binding capacity of serum proteins as a function of pH of the serum or of added calcium ion. The determination of the nortion of calcium which is present in the ionic form in the serum depends on the ORK STOPPER fact that the existing equilibrium between ionic and bound calcium will not be SBMPLE changed by the insignificant weight of calcium added with the Cadsto the serum. TUBE If this condition is met, the ratio of ionic Ca4 to bound Ca" following the rapid exchange will be the same as that for the inactive calcium in the two forms. Thus, rim*. a. Th. Centritus. am- if one ascertains the total umbly w i t h th* Sack SupCa46 in a serum sample, portad by th. Nylon Fa.t*n.d separates the ionic calcium t o th. Cork mtopp..
from the bound, and determines the Ca4 in the ionic fraction, the experiment is completed. This paper describes the apparatus and techniques which can be used to perform this experiment and gives some typical results obtained by the classes studying tracer techniques. THE EXPERIMENT A total of I@-12 mg. of calcium per 100 ml. of serum is present in nonlaying chicken-blood serum. Approximately 50 per cent of this total calcium is bound to the protein fraction and may be designated as nondiffusible calcium. One may visualize an equilibrium existing between W moles of diffusible calcium ion and X moles of nondiffusible calcium bound to protein still having Y available sites for binding. If ionic CaG of high specific activity is added to the serum and the equilibrium is not changed, the Cad' will distribute itself in the ratio of the two forms originally present. If, however, the number of available sites is exceeded by the weight of Ca++ carrier added with the Ca4, a different equilibrium value is established and the experimental result will indicate the per cent ionic calcium present under these new conditions. The radioactive calcium purchased from Oak Ridge National Laboratory is designated as "Ca-45-P-2, High Specific Activity" and has a specific activity of from 5 to 60 millicuries per gram of calcium. The radioisotope is obtained as the chloride in a dilute hydrochloric acid solution. Seamless cellulose dialyzer tubing from Fisher Scientific Co. has a pore size such that ionic calcium can be separated from bound calcium under mild centrifugal conditions to be described later. The a/&-inchtubing was more adaptable' to our equipment than larger sizes. The serum was usually obtained from the blood of nonlaying chickens and used without dilution by a buffer. However, in a few cases it was diluted by the addition of sodium borate buffer and comparable results were obtained. Recently silnilar studies have been carried out with crystallized bovine plasma albumin purchased from Armour Laboratories. The same principles can be illustrated to the class with this protein source. Figures 1and 2 show the special equipment. A piece of nylon cloth about 6 inches square, a 50-ml. conical test tube, a special cork stopper as shown, and a centrifuge complete the items necessary for the separation of ionic calcium from the remainder. Radioactive calcium samples (25 or 50 pl.) of the original and centrifuged fractions are dried on copper plates. Thin-win-
VOLUME 33. NO. 7, JULY, 19%
dow G. M. counter tubes (1.9 mg. per ~ m . with ~ ) associated scalers are used for all countiug determinations. Enough tracer calcinm is added to 5 ml. of chickeublood serum so that a 25 pl. aliquot will record approximately 500 counts per minute under 10 per cent geometry conditions. This will usually correspoud to 1-5 pl. of the Ca" sohtion. Two 25-p1. samples are withdrawn after the solution is thoroughly mixed and prepared for countiug. The method of separation has been reported by Clegg (4) and is a modification of that reported earlier by Benjamin and Hess (6). The sack and centrifuge assembly are prepared as indicated in Figures 1and 2. The solution after sampling is poured into the dialyzing tubing previously tied off a t the bottom and then sealed by knotting the upper end. The corners of the nylon square are collected a t the upper end of the sack and tied securely with a string just below the knot (see X in Figure 1). The string is then passed through the hole in the cork stopper, looped over a short wood plug, returned through the hole, and tied just below the knot in the sack. The nylon must support the dialysis tubing so that it will not rupture. Ceutrifugation time of 15 minutes a t 2000 r.p.m. is necessary to collect about 0.5 ml. Duplicate samples of 25 pl. of the centrifugate are prepared and all samples counted under the same geometry conditions. The per cent ionic calcium is then determined directly from the counting data as per cent Ca++ = c.p.m./25 pl. of Ca" in celtrifngate + c.p.m./25 pl. of Ca4"n original. A variation in the above procedure may be made so that the effect of pH on calcium binding can be measured. This merely requires the adjustment of the pH of the serum with dilute hydrochloric acid after the addition of the Ca45 but prior to the initial sampling. Similarly in the stndy of added calcium versus calcium binding, the addition of inactive calcium is done prior to the initial sampling. All other steps are the same. TYPICAL RESULTS
Effect of pH o n Calcium Binding
8.3 48
pH of serum 3'% Ionic calcium
7.1 55
6.1 77
5.0 93
state and about 45 per cent as ionic calcium in the former case. Table 1 contains some typical student results on the effect of pH on calcium binding. The results of a stndy on the effects of added calcium on per cent ionic calcium are given in Table 2. These studies were done on 5 ml. of cockerel serum a t a pH of 8.2. TABLE 2 Effect of Added Calcium on Calcium Binding i n Cockenl Serum
Cat+ added
(mg./ml.) Ionic calcium 1%)
0.017 0.26 0.52 1 . 2 2 . 4 5 . 2 6 . 9 9 . 1 47
41
4%
64
61
62
63
59
Another group of results from a similar study is given in Table 3. These determinations were also carried out on 5-ml. portions of laying-hen serum a t a pH of 8.2. DISCUSSION
These experiments illustrate a number of chemical principles and a practical use of a radioactive tracer. The rapid exchange and attainment of equilibrium can be readily demonstrated. The experiment demonstrates the advantages of radiometric analysis since the per cent ionic calcium can be determined accurately and rapidly by this method. If the amount of total calcium present is known from other chemical analyses, the concentration expressed as concentration of both bound and ionic calcium can be obtained from the radiometric data. The need for high specific-activity calcium is obvious if one wishes to use the method to determine the correct equilibrium value. The binding capacity of the serum proteins as a function of added calcium is an informative experiment. It is an interesting observation that the protein fraction present in laying-hen serum binds more calcium. Since added calcium remains in the ionic form, this fraction appears to be saturated with respect to calcium.
The rate of exchange of the added ionic Ca" with the protein-bound calcinm is very rapid as would be expected from the nature of the bonds. Hence the time necessary for the added ionic calcium to come to equilibrium with the bound as well as the ionic calcium in the system is also short. In each laboratory session, one group of two students can perform the separation as LITERATURE CITED rapidly as possible (about 10 minutes) and determine MCFARLAND, R. H., AND R. E. HEIN in Use of Imtope8 in the per cent ionic calcium present. Other groups can Plant and Animal Research, TID-5098, 27 (1952). CLEQQ,R. E., AND R. E. HEIN,Pmlt~ySei., 32, 865 (1953). carry nut the experiment under the same conditions exVISEK. W. J., R. A. MONROE, E. W. SWANBON, AND C. L. cept the time of contact of the Ca45with the serum can be CO&, J. airy Sci., 36,373 (1953). longer. The per cent ionic calcium determined was the CLEQQ, R. E., Chemist Analyst, 38, 87 (1949). same regardless of the time allowed for mixing. VariaBENJAMIN. H. R.. AND A. F. HESB.J. Biol. Chem., 100, 27 tions from ten minutes to two hours were tried. From these data, it was assumed that equilibrium was atTABLE 3 tained rapidly (in less than ten minutes). i n Calcium Binding i n LayingThe per cent ionic calcium was usually determined on Effect of Added Calcium hen Serum cockerel serum but occasionally serum from laying hens 0.62 1.24 0.27 0.018 was analyzed in a similar manner. In the latter case Caf+ added (mg./ml.) 89 67 85 Ionic calcium (%) 23 about 20ber cent of the calcium was present in the ionic
