are shown in Table VII. Each sample contained 250 mg. of gelatin dissolved in 25 ml. of water. These were titrated t o p H 11.5 with 0.3000N Ba(OH)2, then excess Ba(OH)2 amounting to 18.000 X equivalent was added to each and the preparations backtitrated to p H 11.5 with 0.2000AT CUSOI. This protein utilizes 1.887 0.025 (S.D) milliequivalents of alkali per gram in excess of that required to neutralize ordinary acidic groups and to account for enolic groups stabilized by reaction with copper. The proteins vary somewhat as to the amount of alkali they can consume in excess of these requirements, but, once this is known, the amount of protein in a sample can be measured by titrating it t o p H 11.5 to neutralize the ordinary acidic groups, adding a known and excessive amount of barium hydroxide, and back-titrating to p H 11.5 with standard copper sulfate. The weight is calculated by finding the difference, in equir-
alents, between the amount of hydroxide used above p H 11.5 and the amount of copper and multiplying this times the appropriate factor. This method should prove useful with protein solutions that are too highly colored or too turbid to be suitable for spectrophotometric measurement.
*
LITERATURE CITED
(1) Block, R. J., Science 108, 608-9
(1948). (2) Block, R. J., Bolling, Diana, “The Amino Acid Composition of Proteins and Foods,” 2nd ed., pp. 3-34, C. C Thomas, Springfield, Ill., 1951. (3) Block, R. J., We,i;s, Kathryn, “Amino Acid Handbook, C. C Thomas, Springfield, Ill., 1956. (4) Datta, S. P., Rabin, B. R., Biochim. et Btophys. Acta 19,572-4 (1956). (5) Dobbie, Hazel, Kermack, W. O., Biochem. J . 59, 257-64 (1955). (6) Freeman, H. C., Smith, J. E. W. L., Taylor, J. C., Nature 184, 707-10 (1959). (7) Gurd, F. R. X., Wilcos, P. E., d d -
vances i n Protein Chem. 11, 311-427
(1956).
(8) H o w , P. E., J.Bid. Chem. 49, 93-107 (1921). (9) Kober, P. A,, Ham, A. B., J . Am. Chem. SOC.38,457-72 (1916).
(10) McDonald, H. J., “Ionography,” p. 36, Yearbook Publishers, Chicago, Ill., 1955. (11) Mehl, J. W.,Pacovska, E., Winzler, R. J., J . Bioi. C h a . 177, 13-21 (1949). (12) Plekhan, M. I., Russianora, N. D., Zhur. ObshcheZ Khim. 23, 512-18 (1953). (13) Spector, W. S., “Handbook of Biological Data,” p. 24, W.B. Saunders, PhiladelDhia. Pa.. 1956. (14) Strichand, R. D., Mack, P. A., Childs, w.A,, ANAL. CHEhi. 32, 430-6 (1960). (15) Tshugaev, L., Ber. deut. chem. Ges. 40,1973-80 (1907). RECEIVED for reviex September 6, 1960. Accepted November 14, 1960. Division of Biological Chemistry, 138th Meeting, ACS, Sew York, K.Y., September 1960. Investigation supported in part by a research grant, H2100, from the National Heart Institute, Sational Institutes of Health, Public Health Service, Department of Health, Education, and Welfare‘
Investigation of Nuclear Fast Red Method of Baar for Direct Spectrophotometric Determination of Calcium in Serum, Urine, and Spinal Fluid G. R. KINGSLEY and OZlE ROBNETT Clinical Biochemistry laboratory, Veterans Administration Center, and Department of Physiological Chemistry, School of Medicine, University o f California, los Angeles 24, Calif. The optimum conditions for the use of purified nuclear fast red (NFR) were investigated for the determinotion of calcium in small amounts of serum, urine, and spinal fluid b y a simple, direct, and rapid (5 to 10 minutes) spectrophotometric method with well known spectrophotometers. The analytical results obtained with this method for the determination of calcium in biological specimens were in good agreement with those of established methods.
A
method for determining calcium in serum and urine which does not require t h e removal of proteins, the precipitation of calcium as oxalate, or a tedious titration procedure, is very desirable as a clinical procedure, especially where speed is desired and large numbers of determinations are made. Calcium methods of this kind have been reported by Kingsley and Robnett (6, 7 ) , Baar (I), and Chilcote and Wasson ( 2 ) . The method of Kingsley and Robnett required a dye, disodium - 1 - hydroxy - 4 - chloro - 2,2DIRECT
552
0
ANALYTICAL CHEMISTRY
diazobenzene - 1,8 - hydroxynaphthalene-3,6-disulfonic acid, which binds calcium t o form a complex with less light absorption in solution than the original dye. This condition requires setting the unreacted dye as a blank a t the greatest absorbance reading permissible for the photometer and then reading the decreasing densities of specimens to measure the amount of calcium present. This situation places a maximum “load” on the electronic system of the photometer which may give nonlinear results if the photometer is not operating efficiently. Although these requirements for proper use of the method have discouraged more general acceptance, this method has been very satisfactory in our hands. Chilcote and Wasson (2) employed ammonium purpurate for spectrophotometric calcium determination, which provided a color that was read in the conventional manner on t h e spectrophotometer. This method, however, has three principal defects: instability of t h e ammonium purpurate standard, greater temperature sensitivity of the color produced, and t h e requirement
that the color be developed a t 4’ to 15’ C. The chloranilic acid calcium method of Ferro and Ham (4, 5 ) is not simple, rapid, or direct, as i t requires a large sample of serum, precipitation of proteins, washing with isopropyl alcohol, centrifugation, resuspension of precipitate, resolution n-ith EDTA, etc. This procedure requires a t least 60 minutes to perform. The method of Baar ( 1 ) in which nuclear fast red (NFR) was used, apparently avoids the objectional features of t h e other direct calcium methods cited above. However, one difficulty is encountered: KFR obtained as a solid from commercial sources requires further purification to remove impurities as described by Baar ( 1 ) before i t is satisfactory for the determination of calcium. The effects of variations of time, temperature, concentration of reagents, interference of nonspecific substances, etc., are evaluated to determine the optimum conditions for the use of NFR. We believe these modifications have improved Baar’s original KFR calcium method considerably.
MATERIALS AND METHODS
Reagents. SUCLEAR FAST RED (NFR) ( 1 ) is purified as follows: Dissolve 5 grams of commercial IYFR in 250 ml. of warm 50y0ethyl alcohol and cool a t 15' C. for 36 hours. Filter on a Buchner funnel, wash the precipitate with ethyl alcohol until washings are colorless, and finally wash with 100 ml. of ether. Dry over phosphorus pentoxide. Unrefined S F R is obtainable from George T. Gurr, Ltd., London, S.K. 6, England, or Borden Chemical Co., 5000 Langdon St., Philadelphia 24, Pa. A purified stabilized stock nuclear fast red solution, Fsst R , ready for use may be obtained from I
