Rapid and Precise Determination of Total Serum Protein by Thermochemical Analysis Earl B. Smith' and Peter W. Carr2 Department of Chemistry, University of Georgia, Athens, Ga. 30602
A thermometric titration for the determination of total serum proteins is reported. The titrant, 12-phosphotungstic acid, reacts stoichiometrically with the protonated basic amino acid residues of a protein. High purity cesium chloride may be used for standardization of the titrant. Both reactions are carried out in 0.1M hydrochloric acid. Results indicate that either a pure protein (bovine serum albumin) or mixture of proteins can be titrated in solutions as dilute as 1 g/l., which is equivalent to a 1.5mM solution of the reactive group, or as concentrated as 15 g/l. Under appropriate conditions, including the rate of addition and stirring, the stoichiometry is independent of protein concentrations and the end point is precise to 0.5%. Interferences from nitrogen bases such as bilirubin at the levels commonly encountered in serum appear to be negligible. At present, the protein content of 1 ml of human serum can be determined with a precision of 0.5%. Correlation of the method with Kjeldahl nitrogen analysis and biuret colorimetry is acceptable.
The need for a convenient. rapid, and precise method for the assay of proteins has been a problem in both clinical and bioanalytical chemistry for some time. At present the rPjcwncc method for proteins is the Kjeldahl nitrogen method which was introduced in 1883 ( I ) and first applied to the analysis of proteins in blood in 1921 ( 2 ) .The technique is precise to better than 1% in the hands of an experienced analyst when the measurement step is a titrimetric assay for ammonia and the semimicro procedure is used ( 3 ) .The method can be automated ( 4 ) and the resultant ammonia determined colorimetrically by Nesselerization ( 5 ) or by the Berthelot color reaction (6). Relative precision under the first conditions is reported to be about 1%( 3 ) .In either case, the analysis time is long due to the required decomposition of the protein to ammonia and subsequent distillation (or diffusional separation) of ammonia ( 7 )from a strongly basic solution. Although the nitrogen content of a protein or proteins in a mixture can be specified with reasonable precision and accuracy. results are often reported as the per cent (w/v) of protein in the sample. For a given pure protein preparation, this procedure will not entail any serious determinate errors since any given protein is a true compound and has a constant elemental composition. However, the nitrogen content of various proteins is different and in fact is widely divergent; e g , a value as high as 30.0% nitrogen has been determined for clupein from her-
ring (8) and a value of 12.6% nitrogen has been found for the human az-zinc glycoprotein (9). The nitrogen content and other chemical characteristics of the major electrophoretic fractions of human serum are summarized in Table I (10-23). The spread of the nitrogen content of the plasma proteins is summarized in Figure 1, curve a. As a consequence of the variability of the nitrogen content of proteins, one must view the measurement of proteins by the Kjeldahl method as being similar to the classical bromine, iodine, or saponification numbers of mixtures rather than a precise determination of the mass of protein in a sample. Henry ( 3 ) reports that the nitrogen content of the major proteins found in human plasma has a range of 8.48 to 17.0% corresponding to nitrogen to protein conversion factors of 12.5 and 5.9, respectively. A value of 6.54, i.e., 15.3% by weight, as an average value has been suggested (24) and used by many clinical chemists as a viable compromise; however, the classical value (6.25) is still more widely used. Therefore, even though the Kjeldahl method is precise it may not be more accurate than 4-5%. There are a very large number of relatively specific colorimetric methods for proteins, each of which is ultimately standardized on the basis of the Kjeldahl nitrogen content of a reference sample. These methods include the biuret reaction (25) (which is closely related to the number of peptide bonds of the protein), direct measurement of the absorbance at 280 nm (26, 27) (which is due to the presence of the aromatic amino acids: tyrosine and tryptophan), and measurement of the absorption at 215 nm (which is due to the peptide bonds) (28, 29). Related methods include binding of anionic dyes to the positively
Present address, Department of Clinical Chemistry, Universit y of M a r y l a n d M e d i c a l School, Baitimore, Md. A u t h o r t o w h o m requests for reprints should be addressed. (1) J. Kjeldahl. Fresenius' Z.Anal. Chem., 22, 366 (1883). (2) P. E. Howe, J. Biol. Chem., 49, 109 (1921). (3) R. J. Henry. "Clinical Chemistry," Harper and Row, New York, N.Y., 1964, pp 175-179. (4) A. Ferrari. Ann. N . Y Acad. Sa..87. 792 (1960). ( 5 ) D. Hunter, Amer. J. Clin. Pathol., 17, 650 (1947). (6) A. L. Chaney and E. P. Marbach. Clin. Chem., 8, 131 (1962). (7) E. R. Tompkinsand P. L. Kirk, J. Biol. Chem., 147, 477 (1942).
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ANALYTICAL CHEMISTRY, VOL. 45, NO.
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T. Ando, S. I. Ishii, and M. Sato. J. Biochem. (Tokyo),
46, 933 (1959). W. Burgi and K. Schmidt, J. Biol. Chem., 236, 1066 (1961). R. D. Strickland, R. A. Mack, F. T. Gurrule. T. R. Podlenski. 0. Salome, and w. A. Childs, Anal. chem., 31, 1410 (1959). A. Razand D. S. Goodman, J. Biol. Chem., 244, 3230 (1969). S. H. Armstrong, Jr., M. J. E. Budka, K . C. Morrison, and J. Hasson, J. Amer. Chem. SOC.,69, 1747 (1947). S. Margolisand R . G. Langdon, J. Biol. Chem., 241, 469 (1969). E. Brand, 6. Kassel, and L. J. Saidel. J. Clin. Invest.. 23, 437 (1944). C. 6. Kasper and H. F. Deutsch, J. Biol. Chem., 238, 2325 (1963). W. C. Parker and A. G. Bearn, J. Exp. Med., 115,'83 (1962). G. R. Tristram and R. H. Smith, Advan. Protein Chem., 18, 227 (1963). G. R. Cooper, "The Plasma Proteins," Voi. 1, F. W. Putnam, Ed., Academic Press, New York. N.Y., 1960, p 51. G. R. Tristram, "The Proteins," Vol. 1, Part A, H. Neurath and K. Bailey. Ed., Academic Press, New York, N.Y., 1953, p 224. W. E. Marshal, J. Biol. Chem., 241, 4731 (1966) R. J. Winzler. "The Plasma Proteins," Vol. 1, F. W. Putnam. Academic Press, New York, N.Y., 1960, p 309. B. M. Kagan. J. Clin. Invest., 17, 369 (1938). 0. Warburg and W. Christial, Biochem. Z.,310, 384 (1941). E. D. C. Chariaviglio, A. V. Wolf, and P. G. Prentis, Amer. J. Clin. Pafh., 39, 42 (1963). A. G. Gornall. C. J. Bardawill. and M. M. Davis, J. Biol. Chem., 177, 751 (1949). T.W. Goodwin and R. A. Morton, Biochem. J., 40, 628 (1946). G. H. Beaven and E. R. Holiday, Advan. Protein Chem., 7, 320 (1952). W. J. Waddell, Lab. Clin. Med., 48, 311 (1956). M. P. Tombs, K. B. Cooke, F. Souther, and N. F. Mclagan in "Protides of Biological Fluids," H. Peters, Ed., Elsevier, Amsterdam, 1960, p 37.
Table I. Chemical Composition of the Plasma Proteins Basic amino Protein w % Of acid residues fraction index Protein total protein (78) % nitrogen per io5 g ... ... 12.9 (27) 1 Haptoglobins 20.1 (70) 118 (77) 0.1-0.5 2 Prealbumin 14.9 (70) 143 (77) 55-64 3 Albumin
y-G lobul ins Transferrin 6 Ceruloplasmin 7 Glycoproteins 8 @-GI0bul ins 9 a-Lipoproteins 10 a,-Globulins 11 y2-Macroglobulins 12 Fibrinogen 1 3 &Lipoprotein 1 4 az-Globulins 15 Orosomucoid 4
5
Total or av
10.1-1 7.2 3-6.5 0.2-0.5 1.2 11.6-15.2 3.4 7.6-10.1 1.5-4.8 3-8.5 9.1 3.6-7.2
0.5 109.90
15.2 (72) 15.4 (27) 13.8 (27) 12.6 (23) 14.8 ( 7 4 ) 8.0 (72)
97 119 98 90
(77) (76) (75) (22)
... ...
...
...
14.8 16.7 (27) 4.2 (72)
125 (79) 95 (73)
...
...
10.1 (27)
73 (20)
13.7O
...
0
0
2
4
6
PROTEIN
Figure 1.
13OC
This is the s u m of the minimum value of all of the above numbers. It should total to loo%, but because of the propagation of analytical errors and the variety of sources of the data, it does not. This is an average of all of the above, weighted in proportion to their weight per cent of total protein. Its inverse is the nitrogen to protein conversion factor. CThisis an averaae value weiahted as in b .
charged sites on the protein (30). Some of these procedures are reasonably specific for one type of protein; for example, the dye 2,4'-(hydroxybenzeneazo) benzoic acid is relatively selective for albumin (31). Notwithstanding, all of these methods are limited to a few per cent precision and are highly subject to interferences. A number of reagents are known to precipitate proteins quite completely from solution. These include tungstic acid, perchloric acid, trichloracetic acid, both phosphomolybdic and phosphotungstic acids (PTA), metaphosphoric acid, as well as several cations such as mercury(II), cadmium(II), iron(III), copper(II), and zinc(II), and miscible organic solvents including acetone, methanol, and ethanol. All of these materials have been used to prepare protein-free filtrates prior to analyses in which proteins are known to interfere. Some of these reactions are stoichiometric and correspond to well-defined chemical processes (32). In essence, the action of anionic precipitant corresponds to reaction of the anion with the protonated protein (H,Pn+) and can be represented in acid media as (1) The number of positively charged sites on a protein is principally determined by three factors: the number and type of the basic amino acids (histidine, lysine, and arginine) in the protein and the pH of the solution. We have shown (33) that 12-phosphotungstic acid reacts with proteins where the stoichiometry corresponds closely to the number of positive charge sites on the molecule (at low pH the stoichiometry is a measure of the sum of the basic amino acids and the N-terminal amino acids) and where the end point in a titration of pure proteins or polypeptides can be located with good precision by constant rate thermometric titration. It is the purpose of the present (30) R. D. Strickland. T. R . Podleski, F. T. Gurule, M . L. Freeman, and W. A. Childs. Anal. Chern., 31, 1403 (1959). (31) D. D. Rutstein. E. F. Ingenito. W. E. Reynolds. and J. M . Burke, J. Clin. Invest.. 33, 211 (1954). (32) L. Silverman, B. Schreiner, and D. Glick, J. Cell Biol., 41, 768 (1969). (33) E. B. Smith, Ph.D. Dissertation, University of Georgia, 1972.
I
1
8
10
I, 12
14
FRAC~~ON INDEX
Chemical composition of plasma proteins relative to
albumin The protein fraction index lists the proteins in their order of appearance in Table I : curve a, nitrogen content ( 0 ) ;curve b, basic amtno acid content ( A )
work to extend that method (3.3) to the analysis of the protein mixtures found in human serum and evaluate the possibility of interfering species. The method described here is similar in many respects to the Kjeldahl nitrogen assay; however, the procedure reported here measures the number of moles of basic amino acid r m i d u c s present in a protein mixture. As the data of Figure 1 suggest: there is no complete correlation between the contribution a given protein makes to the total amount of nitrogen in the mixture and the total number of basic amino acids of serum. Consequently, an exact correlation between the Kjeldahl nitrogen assay and equivalents of basic amino acids is not expected. In light of the preceding discussion concerning the absolute accuracy of the analysis of protein mixtures, we believe that the thermometric titration of proteins may prove to be under certain conditions a reasonable complement and/or alternative to the Kjeldahl nitrogen analysis of proteins.
EXPERIMENTAL Apparatus. All thermometric titrations described in this work were carried out in a Dewar flask and air bath described in a previous communication ( 3 4 ) . Temperature changes were monitored with a n ac Wheatstone bridge in conjunction with the phase-lock amplifier described previously (35). Titrant solutions were added via a syringe drive pump. In most of the work reported here, a precision syringe or gas tight syringe was used to prevent seepage and concomitant evaporation of the very concentrated titrant solutions. The syringes were driven by a syringe pump (Sage, Model 234-3) and pumped through very narrow h e Te!lon tubing (Hamilton Company, KF28TF). The narrow tubing employed was crimped to minimize creepage 01 the cesium phosphotungstate precipitate. The Dewar flask contents were stirred with a four-blade stirrer. Each blade was circular, had a diameter of 0.4 in., and was pitched a t an angle of +:HI" from the stirrer's shaft. An Inframo (Model RZR1-64) motor was used to rotate the stirrer a t speeds varying from 50 to 2500 rpm. The titration curves were recorded on a Hewlett-Packard X-Y recorder (Model 7000A), the x-axis being driven by the voltage generated from a linear position transducer which tracked the syringe drive pump ( 3 5 ) . The use of a linear position transducer was deemed necessary because of the inability of the pump t o maintain constant rate with the highly viscous titrant solutions. The biuret determinations were carried out with a Cary Model 15 YV-visible spectrophotometer (34) P. W. Carr, Therrnochirn. Acta, 2, 505 (1971). (35) E. E. Smith, C. E. Barnes, and P. W. Carr. Anal. Chern.. 44, 1663 (1972).
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 9. AUGUST 1973
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r
C
solution which was cleared by centrifugation and decantation of the supernatant fluid. The titer of PTA against cesium chloride was stable up to one month or longer even though a slight blue color developed after about two weeks.
/
/
r
with matched 1-cm cells in the reference and sample beam. Gornall's (36) method was used to develop the color. An ordinary clinical centrifuge (International Equipment Company, Serial 248) or a refrigerated unit (Sorvall, Model RC2-B) was used to separate the blood cells when plasma was required or both cells and fibrin when serum was used. The clinical centrifuge was also used in removing the suspended particulates which were always present in freshly prepared concentrated PTA solutions. Plasma was clotted by addition of calcium chloride. Reagents. Bovine serum albumin (Fraction V, powder) was obtained from Schwarz/Mann and according to their certificate of analysis was 97% albumin, the remainder being globulins. All calculations were based on a n assumed purity of 100%. Any correction for purity would be somewhat ambiguous; a t most our absolute accuracy will be in error by 3%. The 12-phosphotungstic acid was Baker analyzed reagent grade and was used after removal of insoluble matter from the solution. Cesium chloride (99.96%), used for the standardization of the titrant, was obtained from Fisher Scientific. It was dried a t 110 "C for 24 hr and stored in a desiccator. All other reagents were analytical grade. Procedure: Concentrated stock solutions of the purified proteins were prepared by carefully dissolving the desired quantity of protein in a deficient amount of cold water by stirring on a cold plate stirrer (Stir Kool, Model SK-12, Thermoelectronics Unlimited). Once dissolved, the solution was quantitatively transferred to a volumetric flask (rinsing with cold water). The final stock solution was stored in an ice bath until used. All titrations were carried out on a total volume of 25 or 30 ml of solution by first pipetting the desired quantity of stock solution with a "to contain" pipet which was then rinsed with distilled water into a Dewar flask; a sufficient volume of hydrochloric acid and distilled water was then added to adjust both the volume and acid concentration to the desired value (normally O.1M). The Dewar flask was rinsed with alcoholic potassium hydroxide immediately after each run. This type of procedure provided maximum reproducibility. Prior to titration, the titrant was separated from the analyte solution by a small air bubble which was introduced into the tip of the reagent delivery line. About 2 min were allowed for the contents of the Dewar flask to reach temperature equilibrium before the titration branch was recorded. During this time, the temperature bridge was nulled and any temperature shift indicated on the recorder was compensated for by appropriate adjustment of the associated ramp generator. The reagent delivery rate was calibrated by titrating a 25-ml aliquot of a solution of cesium chloride of known concentration. Blood samples for the determination of protein were collected the previous day from hospital patients. These samples. amounting to about 15 ml each, were allowed to clot and then separated by centrifugation for 10 min at 15,000 rpm and 4 "C. The serum was decanted and stored a t 4 "C until ready for use. The reagent, 12-phosphotungstic acid, was prepared by weighing out the desired amount of reagent grade material and dissolving it in deionized, glass distilled water. This resulted in a cloudy
RESULTS Preliminary Experiments. The thermometric titration of proteins with many of the common anionic and cationic precipitants was studied in a number of preliminary experiments. The reagents tested were sodium tungstate, trichloracetic acid, perchloric acid, sodium tetraphenylborate, lead(II), silver(I), mercury(II), cadmium(II), iron(ID), tetraphenylstibonium, and tetraphenylarsonium. All of these reagents did form insoluble protein precipitates under appropriate conditions, i. e., acidic solutions for the anionic reagents and basic solutions for the cationic precipitants. In every case, an ill-defined thermometric titration curve was obtained that was attributable to either a slow reaction or to a very small enthalpy change. The most promising reagent tested in preliminary work was lithium reineckate which produced well-defined rectilinear titration curves. A more detailed investigation via colorimetry and gravimetry indicated that the precipitation process is very incomplete except when an excess of the reagent is present. Prior to reaching the titration end point, a soluble complex between the protein and the anion is formed. After the end point, virtually all of the protein is precipitated. As a consequence of the complex chemistry, the thermometric end points were precise to only 2-5%. The next reagents tested were 12-phosphomolybdic acid and 12-phosphotungstic acid (F'TA). Both of these materials produced thermometric titration curves which seemed to be analytically useful and both reagents have been used as protein precipitants (32). Of the two materials, 12phosphotungstic acid gave more nearly linear curves (see Figure 2) and better end-point precision (vide infra). Several preliminary experiments on the precision of end-point location in the titration of bovine serum albumin indicated that this reagent was suitable for further investigations. Titrant Standardization. Approximately 350 g of reagent grade (highly hydrated) 12-phosphotungstic acid will dissolve in water to produce a final volume of 100 ml of solution. The solutions proved to be quite stable over a period of more than a month. It has been determined (37) that the solubility of PTA expressed in the unhydrated form, H3PW12040, is 176.3 g/100 ml of solution. We have found that maximum precision is obtained when the titrant contains no more than 130 g/100 ml of 12-phosphotungstic acid. This concentration of titrant produced the most reproducible protein titration curves but had little influence on the precision of cesium titrations. Because of the extremely hydrated state of the commercial reagent, it is impossible to prepare a standard solution of PTA by weighing. It was therefore necessary to develop a standardization procedure. The fact that the anion decomposes in neutral and basic solutions precludes standardization by titration with strong base. The PTA anion is easily reduced a t the DME (38). There are several waves starting at about -0.2 V us. the SCE in 0.1M sulfuric acid. Nevertheless, controlled potential electrolysis, in the above medium, on a mercury pool at a potential of -0.32 V L'S. the SCE was precise to only 5%. The current was not mass transfer controlled and decreased very slowly toward zero. Consequently this method was abandoned.
(36) W. R. Faulkner, "Methods of Clinical Laboratory Procedures," W. R. Faulkner. and J. W. King, E d . , Chemical Rubber Publishing Go., Cleveland, Ohio 1970, p 78.
(37) V. Kourirn,J. Inorg. Nucl. Chem., 12,370 (1961). (38) J. H. Kennedy, J. Amer. Chem. Soc., 82,2701 (1960)
B. C.
1
1
,
I
,
I
,
I
I
I
24 5 50.5 85.2
2 4 10
A
,
I
,
,
1
,
TIME OR VOLUME
Figure 2.
Thermometric titration of 30 ml of bovine serum albumin with 0.34N PTA Curve a-protein concentration, 5 g/l.; y axis, 2 m°C/in.; x axis, 16.6 seclin. or 72.2 fil/in. Curve b-protein concentration, 10 g/l.; y axis, 4 rn"C/in.; x axis, 33.2 sec/in. or 144 pl/in. Curve c-protein concentration, 20 g/l,: y axis, 10 m"C/in.; x axis, 33.2 sec/in. or 253 ol/in.
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ANALYTICAL CHEMISTRY, VOL. 45,
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t
Table II. Effect of Cesium Concentration on Analytical Precision and Accuracy Cesium concn, m M
mmol taken
mmol found
Yo ud
4.7a9a 0.1439 0.1429 0.4 9.950~ 0.2985 0.2974 0.8 0.6009 0.5992 0.5 20.030 1.198 0.2 40.03b 1.20, 1.796 1.796' 0.8 59.85' "The titrations were carried out in 30 mi of 0.10M hydrochloric acid at a rate of addition of 2.31 pequiv/sec. The titrant is approximately 1.3N phosphotungstic acid, *Rate of addition changed to 5.76 pequiv/sec. Ail other tactors are as in a . Because of titrant inhomogeneity, we assumed accuracy at this level. Ail preceding results are calculated on this basis. Kinetic and equilibrium curvature should be minimal at this level. Coefficient of variation based upon three replicate determinations.
Table 111. Effect of Protein Concentration on the Reaction Stoichiometry and Precisiona BSA concn taken, g/l.
Titratable groups BSA concnbtaken, found per 100,000 g of proteinC mequiv/l.
D
/---
%
CURVE A.
E. C. D.
ORDINATE LmaC/inch) 1.0 2.5 5.0 25.0
ABSCISSA
(peq. Inch) 9.71 19.5 48 0 I95
T I M E OR VOLUME Figure 3. Thermometric titration curves of 30 ml of cesium chloride in 0.1M hydrochloric acid with '1.32N phosphotungstic acid
ud
0.50
0.735 164.4 2.2 1.47 146.6 1.5 1 .oo 2.94 145.2e 0.4 2.00 5.00 7.35 144.0e 0.8 10.00 14.7 145.ge 0.6 20.00 29.4 154.6 1.8 "The sample protein is bovine serum albumin in a volume of 30 ml of 0.1M hydrochloric acid; the rate of titrant addition is 1.41 pequiv/sec. bTheoretical value computed from the amino acid analysis of the pure protein and the concentration in grams per liter. Direct amino acid anaiysis indicates a value of 147 basic residues per 100,000 g. This estimate is based upon standardization of phosphotungstic acid by the titration of cesium. The theoretical value from amino acid analysis is 147 (37). Based on a minimum of 3 replicates, the mean precision from 1.0 to 10.0 g/l. is 0.8%. e An F test indicates no statistically significant effect of this range of concentration on the stoichiometry at the 95% certainty level.
A unique X-ray method for the determination of heavy metals such as tungsten has been developed by McCall et al. (39). Since very high purity tungsten compounds in the unhydrated form, e . g . , tungstic acid, are available, an analysis for the tungsten content of PTA was developed. This method of standardization was studied with some success; i . e . , the precision of the method was such that a difference of 1 mol 7'0 in concentration could be detected, and the technique was used to survey several commercial sources of PTA. We believed that this method of standardization might not reflect the reactivity of the material under the conditions of its use and furthermore is not generally available to all analysts; hence it was not employed. It is well known (40) that common univalent cations such as potassium, rubidium, cesium, thallium(I), silver(I), and ammonium ions precipitate both phosphomolybdic acid and phosphotungstic acid; indeed an analysis for cesium has been developed based upon the molybdenum reagent (41). The thermometric titration of each of these cations with PTA was studied in 0.1M hydrochloric acid. The solubility product and kinetics of the reaction with cesium chloride were respectively sufficiently small and rapid to produce rather well-defined titration curves. Typical titrations a t several concentrations are shown in Figure 3 and the analytical results as a function of concentration are summarized in Table 11. We therefore thought that it might be possible to determine the number of positive charge sites on a protein by standardizing PTA against cesium chloride and then titrating the protein. In essence, reaction 2 below was carried out and the (39) J. M. McCall, D. E. Leyden, and C. W. Blount, Anal. Chern., 43, 1324 (1971). (40) F. A. Cotton and G. F. Wiikinson, "Advanced inorganic Chemistry," 2nd ed, Wiley. New York, N . Y . , 1966, p 942. (41) C. J. Coetzee and A. J. Basson. Anal. Chim. Acta, 56, 321 (1971).
Curve a-cesium chloride concentration, 2.03mM; y axis, 1 m"C/in.; x axis, 4.15 sec/in. or 7.35 pl/in, Curve b-cesium chloride COnCentration, 4.79mM, y axis 2.5 m"C/in.; x axis, 8.31 sec/ln. or 14.4 $/in. Curve c-ceslum chloride concentration, 9.95mM; y axis, 5 m"C/in.; x axis, 20.7 sec/in. or 36.1 pl/in. Curve d-Cesium chloride concentration, 40.00mM: y axis, 25 m"C/in.; x axis, 33.2 sec/in. or 144 pi/in.
volume of titrant equivalent to the amount of cesium (in moles) was determined. We assumed a 3: 1 stoichiometry for the reaction
+
*
3Cs' PTA3Cs,PTA(+) since there is no accurate means for preparing a standard solution of PTA by direct weight. Protein solutions were then titrated assuming that the reactivity of FTA would remain constant; i . e . , 1 mol of PTA will precipitate 3 mol of cations regardless of the source. This hypothesis was verified by titrating a number of pure proteins and comparing the stoichiometry to the known amino acid composition. The above results (Figure 2) and the data of Table I11 demonstrate that the procedure is valid within experimental error. This method of standardization proved to be rapid, reliable, and sufficiently precise to be used as a basis for subsequent analyses. All protein determinations presented here are based upon the standardization of PTA in terms of moles of precipitable univalent cation per liter and are traceable to a high purity preparation of cesium chloride. When both the PTA and cesium solutions have been preadjusted to a p H greater than 3, the reagent does not precipitate cesium, indicating a change in the nature of PTA at this pH. The acid-base properties of PTA have been examined (42) and its aqueous solution contains between three and four rapidly titratable hydrogen ions per molecule. The nonintegral number of titratable hydrogen ions has been ascribed to a rapid but not instantaneous decomposition of the molecule. Apparently, the decomposition product does not precipitate cesium. Titration of a Pure Protein. Bovine serum albumin (BSA) was chosen as a model protein for the initial work because large amounts are available in high purity (97%) and its amino acid composition and molecular weight are known. The reaction stoichiometry and the analytical precision of the thermometric titration reaction
H,P"+
+ $PTA3-
==F
HnP(PTA)n/3(C)
(3)
were studied a t pH 1 (in 0.1M hydrochloric acid) as a (42) C. Schwarzenback, G. Geier, and J. Sittler, Helv. Chim. Acta., 45, 2601 (1962).
ANALYTlCAL CHEMISTRY, VOL. 45, NO. 9 , AUGUST 1973
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Table IV. Effect of the Rate of Titrant Addition on the Apparent Reaction Stoichiometry a
Rate of addition,a pequiv of PTA/sec
BSA concn, mequiv/l.
Titratable groups found per 100,000 g
% rangeb
0.56 1.250 144.3 1.41 1.250 154.5 0.56 6.321 144.5 1.41 6.321 145.3 3.06 6.321 149.2 7.66 6.321 151.6 a All other reaction conditions are identical to those in Table tive range of duplicate measurements.
T I M E OR
Figure 4.
VOLUME
Effect of stirring on the shape of the titration curve
Protein concentration, 3 g/l.; 30 ml of 0.1M hydrochloric acid; rate of addition, 0.7 pequiv/sec; titrant concentration, 0.8N;y axis 1 m"C/in.; x axis 16.6 sec/in.: curve a-stirring rate, 125 rpm; curve b-stirring rate, 750 rpm; curve c-stirring rate, 2250 rpm
function of protein concentration. These results are summarized in Table I11 and Figure 2. This figure indicates that the titration curves are well defined a t concentrations of 1 to 5 g of protein/l. At higher concentrations, the curve tends to be convex upward in the region of the first break. This behavior is due to the increased viscosity of the precipitate slurry as more and more protein reacts; consequently, the heat due to stirring increases. Evidence for this phenomena is presented in Figure 4. The post titration curve has a much greater slope than the fore titration branch even though the temperature of both the reagent and sample are fairly well matched. Further evidence is noted in that the difference in the slopes of the initial and final branches of the titration curve is a function of the stirring rate, i.e., the difference in the slopes decreases as the stirring rate decreases (see Figure 4, curves a-c). We have observed this phenomenon in other thermometric titrations, all of which involve precipitate formation; none has been as extreme as in the present case. Since the rates of precipitation reactions are often governed by the area of the precipitate, the contents of the titration cell must be stirred vigorously; nevertheless, excessive stirring introduces extreme "viscous" curvature. Consequently, we have empirically chosen a rate of 500 to 1000 rpm as optimal. As Table 111 indicates, the stoichiometry increases a t low protein concentration. We believe this is due to a decrease in the net rate of the chemical reaction between the protein and PTA. Carr and Jordan (43) have shown that spuriously long, i. e., nonstoichiometric, end points will be observed when the rate of a chemical reaction is comparable to or less than the rate at which reagent is added. Table IV summarizes the effect of the rate of titrant addition and concentration of protein on the observed stoichiometry. At low rates of addition, constant stoichiometry is observed but, a t higher rates, the end point error becomes progressively more positive. Similarly, the apparent stoichiometry increases at low protein concentration. This phenomenon also occurs in the titration of cesium a t low concentrations. For this reason, all subsequent analyses were carried out at protein concentrations greater than 2 g/l. and rates of addition less than or equal to 1.4 pequiv/sec. The results indicate that a t concentrations between 1 and 10 g of protein/l. the determination can be carried out with a precision of 0.3 to 0.8%. (43) P. W. Carr and J. Jordan, "Analytical Calorimetry," R. S.Porter and J. F. Johnson, Ed., Plenum Press, New York, N.Y., 1968, p 203.
1692
0.3 0.3
0.0 0.3
0.8 0.7 Ill.
Rela-
Determination of Total Serum Protein. The applicability of the method to serum samples was tested and the results are presented in Figure 5 and Tables V and VI. Figure 5 indicates that the thermometric titration of a protein mixture is quite similar to that of a pure protein even though serum is a mixture of a large variety of proteins. This result implies that the reaction enthalpy for the precipitation of each protein is constant and/or that the free energies of precipitation of each protein are similar. This is a fortunate circumstance in that fractional precipitation and nonequality of reaction enthalpies would probably yield a temperature-volume curve (enthalpogram) with continuous curvature and concomitantly an indistinct end point. Since the fundamental process responsible for the thermometric titration curve is a precipitation reaction and the sample is a mixture of proteins, there exists a possibility that the amount of titrant consumed per unit mass (or volume) of sample might be variable, i. e . , the solubility product of those proteins normally present in low concentrations might not be exceeded in a highly diluted sample. The data of Table IV show that this is not the case, the reaction stoichiometry is essentially invariant over a sevenfold range of dilution. The precision as indicated by the range of duplicate determinations has an average value of 0.5%. Table V presents the results of a more intense study of the reproducibility of the method. A set of ten individual 1-ml samples of serum and a serum pool were analyzed. The individual samples were analyzed in duplicate and the reproducibility measured as the difference divided by the mean of the two runs had an average value of 0.3%. Ten replicate analyses of the serum pool yielded a coefficient of variation of 0.5%. The thermometric titration of serum proteins with PTA evidently has sufficiently high precision to compete with the precision of the semimicro Kjeldahl procedure for proteins. Correlation with Kjeldahl and Biuret Methods. As indicated previously, there should be some tendency for the method described in this work to correlate with other techniques of protein analysis. This hypothesis was tested by performing the semimicro Kjeldahl analysis, a biuret test, and a thermometric titration each in duplicate on ten samples. No attempt was made to establish a normal range or to correlate results with any possible pathology. The results are presented in Figure 6. Correlation coefficients of 0.982 and 0.990 a t the 95% confidence level were obtained for the Kjeldahl and biuret results, respectively. The "I-bars" on this figure were drawn to indicate the range of duplicate measurements of both the y and x axes. They indicate that most of the points fall on the line and the greatest contribution to the variances are in the Kjeldah1 and biuret analyses rather than in the thermometric titration. Some of the points do not fall on the line and are well outside the precision of the techniques. This
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
Table V. Estimate of Precision on Duplicate Analysis on Serum Proteinsa Sample no.
mequiv foundb
% rangeC
99.4 0.2 1 90.2 0.1 2 81.6 2.2d 3 95.9 0.4 4 90.5 0.2 5 90.4 0.2 6 83.8 0.6 7 76.7 0.3 8 85.4 0.3 9 10 75.0 0.8 AV ... 0.34e =Ten individual 1-ml samples of human serum diluted to 30 ml with 0.1M hydrochloric acid, the rate of titrant addition was 1.26 pequiv/sec. *Amount of protein found in units of equivalents of reactive units per 1 ml of sample serum as based on the cesium standardization of the titrant. Range relative to the protein concentration determined in duplicate runs. This value was rejected and not used in establishing the average range of duplicates on the basis of a t test at the 99% confidence interval. e Average of the above results. Ten replicate analyses of a serum pool yielded a coefficient of variation of 0.53%.
Table VI. Effect of Protein Concentration on the Accuracy and Precision of the Analysisa mequiv/g YOrangeC Amounts taken, g mequiv foundb 0.09430 0.3 1.o 0.09417 0.09595 0.3 0.09512 0.3 AV 0.09487e 0.5d =The indicated sample was taken by weight from a serum pool and diluted to 30 mi with 0.1M hydrochloric acid. The rate of titrant addition Based on cesium standardization. Range of was 1.26 pequiv/sec. duplicate determinations. Average of the above ranges. e The computed coefficient of variation is 0.78%. An F test against the average range of duplicate determinations indicates no effect of concentration at the 95% confidence interval. 0.4697 0.91 00 1.934 3.492
A
CURVE
'/ I
ORDINATE (m"C/inch )
0.35 0.50
A.
B. C. ,
,
TlME OR
,
D.
ABSCiSSA (ueq. / Inch)
,
,
2.33
I .o
467 11.6
4.0
46.7
VOLUME
Figure 5. Thermometric titration of h u m a n serum with 0.648N phosphotungstic acid in 3 0 ml of 0.1 M hydrochloric acid Curve a-amount of serum taken, 0.2336 g; y axis, 0.35 m"C/in.; x axis, 1.66 sec/in. or 3.60 pl/in. Curve b-amount of serum taken, 0.4679g; y axis, 0.5 m"C/in.; x axis, 3.32sec/in. or 7.21 pl/in. Curve c-amount of serum taken, 0.9100g; y axis, 1.0 m°C/in.; x axis, 8.31 sec/in. or 18.0 pl/in. Curve d-amount of serum taken, 3.492 g; y axis, 4.0 m"C/in.; x axis, 33.2sec/in. or 72.1 pl/in.
0.443 0.0857 0.1856 0.3322
-87
-83
-
9 m
(44) R. Richterich, "Clinical Chemistry, Theory and Practice," English translation by S. Ramond and J. H. Wilkinson. Academic Press, New York, N.Y., 1969,p 246.
75-
-75
-
-
0
-67
a
63-
-63
A
.
.
7
0
-
I
w 7 55-
r 51'
2
1
-59 0 0
59-
1
Y
E
z
67-
a a
: -I
-71
71-
+ 0 Lz
could be due to an abnormally high or low albumin to globulin (A/G) ratio, i.e., the two largest electrophoretic fractions of serum. Sufficient quantities of the above samples were not available to test this hypothesis. It should be clear from the results presented in Table I that an abnormal value of the A / G ratio will also invalidate the classic conversion factor of 6.25 g of protein/g of nitrogen. It is important to note that both the Kjeldahl and biuret plots extrapolate to a negative intercept of 1.6 g % (or 20mM precipitable cation) on the ordinate axis indicating that the thermometric method may be measuring material which went undetected by both alternate procedures. This result may or may not be real since the extrapolation to zero on the n axis is rather far removed from the actual data points taken. Alternatively any concavity in the initial region of the plot would result in a negative intercept. Since the protein content as measured by Kjeldahl was calculated using the classical factor of 6.25 and that measured by the biuret was calculated using the literature value of 2.77 ( 4 4 ) for the absorptivity of the copper-peptide bond complex, all three determinations are absolute methods of protein determination; L e . , they are independent methods based on conversion factors taken from the literature or traceable to a high purity standard (cesium chloride). Taking these factors into account, some curvature is possible. Due to the scarcity of such samples, data points below 5 g of serum protein/100 ml were not obtained and this hypothesis could not be tested.
-79 4 m
5
w
a
79-
\
-
mc
-55
6
3 :
1 ' 74
' 7 8 ' 8 2 ' 66 ' 90 ' 9 4 ' 9 8 ' 10.2
'
"I
106' I i O '
T H E R M O M E T R I C PROTEIN [ m e q / m l ) x io2 Figure 6. Correlation of thermometric analysis of total serum
protein with Kjeldahl nitrogen and biuret colorimetry The " I " bars have been drawn to represent the range of duplicate determinations in both methods: a, Kjeldahl analysis ( A ) ; b, Biuret analysis
(0)
Interference Studies. Any determinate errors involved in the comparative analysis such as invalid reagent blanks, standardization errors, etc. might also manifest themselves as a nonzero intercept; however, in our opinion, it would be most surprising if these determinate errors were common t o both the Kjeldahl and biuret analyses. It is possible that the negative intercept indicates that the thermometric procedure is sensitive to some species undetected by the other two methods; i . e . , there is a major interference present in the technique. Since many cationic materials are known to precipitate PTA, tests for interferences were conducted. This was carried out in two different ways. First, serum does contain species other than proteins that may precipitate PTA. The results of Table VI indicate that the reaction stoichiometry is independent of dilution. Thus, these species are either absent or, over the range of the dilutions tested, the solubility products are always exceeded. If such a species were pres-
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9. AUGUST 1973
*
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Table V I I . Interference Study
Material added
Bilirubin
Potassium Alanine Cystine Glutamine Arginine Histidine Lysine Proline Arginine Potassium Calcium Potassium and calcium
Highest normal value, mg/ 100 mla
14.0 4.lC 7.6 5.0 9.7 5.4 3.8 5.8 5.7 5.4 4.lC 5.OC
..,
Added concn, mg/100 mlb
5.0 3OC 30 30 30 30 30 30 30 300 25 0.6C3d
25 ( K + ) ; 0.6(Ca2i)C3d
%change in titer
0.2 0.1 0.1 0.0 -0.5
-0.3 0.0 0.1 0.1 5.3 4.1 0.0
3.4
=According to ref 37. In the serum sample before dilution with 30 ml of 0.1M hydrochloric acid. In millequivalents per liter. Actual concentration in 30 ml of 0.3% bovine albumin: 0.1M hvdrochloric acid.
ent a t a concentration where it only partially precipitates, the reaction stoichiometry would rise as the serum concentration is increased. The possibility of interference from certain species was directly tested by adding them to a serum pool and determining whether the titer increased. Among those species normally found in serum and which potentially precipitate PTA a t pH 1 are bilirubin, ammonium ion, and the amino acids. Other cations such as calcium, magnesium, and sodium do not precipitate PTA and even though they are present in serum, there is little threat of a simple interference. Table VI1 lists the above potential interferences and the maximum amount normally found in human plasma or serum ( 4 5 ) . Ammonium ion is not listed since a concentration of three orders of magnitude higher than that normally present in serum is not sufficient to precipitate PTA. Other amino acids are also present in serum and plasma; however, we arbitrarily chose only those which were present a t a concentration greater than 5 mg/100 ml. As can be seen from Table VII, none of these amino acids produced an increase in titer a t a concentration, in serum, of 30 mg/100 ml, a factor of three- to sixfold above the highest normal value. In order to demonstrate whether amino acids at any concentration could produce an interference, arginine was tested at the 300 mg/100 ml level. This results in only a 6% increase in stoichiometry. Potassium, a t the 5mM level, and a serum sample, which was saturated with bilirubin, gave no increase in titer. At higher concentrations, potassium will precipitate PTA; consequently, potassium oxalate and citrate, which are frequently used as anticoagulants in blood collection, should be avoided and the sodium salts used instead. Although calcium does not precipitate PTA, it does bind very strongly to proteins, particularly to albumin under physiological conditions; consequently, interference by this species was tested by adding calcium to a 0.3% solution of bovine serum albumin. There was no apparent change in titer. A last possibility for interference is the formation of a mixed salt. Consequently, potassium, a t a concentration five times greater than that which would be obtained if all blood cells were ruptured, and calcium, at a concentration four times greater than that found in serum, were added. The simultaneous effect of both potassium and calcium (45) R. L. Searcy, "Diagnostic Biochemistry," McGraw Hill, New York. N.Y., 1969, pp 50,61,113, and 429.
1694
was equivalent to that of 25mM potassium alone. Although this is not conclusive proof that serum contains no interfering substances, we feel that the 30-fold dilution of the serum specimen prior to the titration, as required by the method, eliminates the possibility of stoichiometric precipitation of virtually all reactive species and that a very large excess of PTA would be required before the solubility product of the interfering species, which precipitate PTA, would be exceeded. Such interferences, when and if they do occur, may take place by an occlusion process rather than by true precipitation, and the extent of the interference would be governed by the kinetics of the redissolution of the protein.
CONCLUSIONS We have shown (33) that the reaction stoichiometry between 12-phosphotungstic acid and several purified proteins and polypeptides is dictated by the number of basic amino acids and the solution pH. The location of the first break in each titration curve (see Figures 2, 4, and 5 ) was attributed to the reaction H,PCl,'"-"'+
n - x PTA+ 3
f-
=
The second and final end point corresponds to reaction 5 below and is used in this work as a basis for the determination of total protein.
n - x H,PCl,(PTA)T
x + jPTA3-
===
Since the thermometric titration curves of mixtures and pure proteins are qualitatively similar, each protein fraction of human serum undergoes both the overall and intermediate reactions. The thermodynamic parameters of the various proteins are so similar that PTA indiscriminately precipitates all the proteins in a mixture. This may not be the case at higher pH where there are differences in the acid-base characteristics of proteins; however, this factor was not examined in this work. We have shown that cesium chloride can serve as a practical, although theoretically not ideal, standard for the method. As indicated above, at low analyte concentration or high rates of addition, kinetic effects become important. In principle, this factor will not be identical for both proteins and cesium; in order to eliminate it completely, a highly purified protein standard is needed whose basic amino acid content is known with a greater degree of accuracy and precision than that afforded by the present procedure. A secondary complication is the effect of stirring on the shape and apparent stoichiometry. In the optimum range, this effect is small (1-270); however, the titrations are precise (0.5%) over a wider range of conditions (see Table IV). Nonetheless, the exact stoichiometry is obscured by these processes. Although the present technique measures the total moles of basic amino acids and N-terminal chains contained in the serum proteins, results must be reported as per cent by weight protein in the sample. A similar situation exists with the Kjeldahl determination of total p:otein. The literature data presented in Table I do not clearly indicate whether Kjeldahl nitrogen or the present procedure is a more appropriate measure of total protein. A more rigorous comparison is warranted and is presented in Figure 1.
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 9 , AUGUST 1973
An explicit relationship between the nitrogen analysis result ( K , in grams of nitrogen) and moles of basic amino acid (MBA, in equivalents) and the total weight of a protein mixture (W, g) can be written
K
=
WZ%N,f,
nif i MBA = WZMW,
(7)
where f l , %Nl, n,, and MW, are the fraction of the total mass of protein present as protein i, the per cent nitrogen by weight of that fraction, the number of basic amino acids present in a mole of the protein, and the proteins' molecular weight, respectively. As written, it is clear that a n exact correlation between K and W or between MBA and W, for a series of samples, will occur only under two conditions. First, if all %Tu', or nl/MU', are equal for the various proteins. The data of Table I indicate that this is not the case. Second, exact correlation will occur if the f, for each protein in the mixture is precisely constant for all samples under consideration. This is only approximately true for a set of samples drawn from a number of normal patients, but will frequently be invalid for samples with an abnormal albumin to globulin ratio. The correlation between the measured variables, K and MBA and W will be fair to good if one protein dominates the measurement. This is the case with serum samples where the albumin fraction contains almost 60% of the total mass of protein. In Figure 1, the data of Table I are normalized to that of albumin which is the major serum protein. The dispersion in nitrogen and basic amino acid content is so wide that there does not appear to be any theoretical basis for preferring one or the other technique. A final test of the relative merits of each method for measuring total protein was attempted by computing the correlation coefficient between the average weight of each protein in a sample and the contribution of that fraction to either total protein nitrogen or total protein basic amino acid. This is not an exact test for the desired correlation (Equations 6 and 7). Both correlation coefficients proved to be greater than 0.99 at the 95% confidence interval, this result being dictated by the dominance of the albumin fraction. A statistically significant intercept was obtained in each case. Since there does not appear to be any theoretical justification for preferring one technique over the other, at least for the serum proteins, some empirical comparisons should be made. Implementation of Kjeldahl analysis by colorimetry or titration does not seriously influence the total time of analysis since the slow step is the decomposition of protein. The actual analysis time for the thermometric analysis is quite low. After pipetting the sample and the requisite quantity of acid, a complete titration curve is obtained in 10 min. If several Dewar flasks are available, only a few seconds are lost in cleaning the flask.
The precision of the semimicro Kjeldahl method and the thermometric titration are comparable 0.5-1.0% us. 0.30.8% in the optimum range, respectively (see Table V). A recent report (46) and previous experience have shown that a t sufficiently high concentration the precision of thermometric titrations can be improved by pretitrating the sample with a high precisipn buret or pipet. This is due to the fact that the syringe drive pumps used in this work are, a t best, precise to 0.2% (35). At present the precision of routine clinical procedures for total protein is only marginally capable of discerning differences in a healthy and demographically homogeneous population, but the methods are incapable of measuring intrapersonal variations with time (47). The data presented in Table V and estimates of intrapersonal variations (47) indicate that the present method can detect changes in total protein in the above population. A major drawback of the present procedure is the large volume of sample (1 cm3 of serum or about 60 mg of protein) required for analysis. This is not a problem in Kjeldah1 analysis, particularly if colorimetric estimation of ammonia is used. The protein content of cerebrospinal fluid is 15-45 mg/100 ml (48) and cannot be measured by the titration method without preconcentration. For routine clinical work, a microtitration procedure employing a 3-ml Dewar flask is being developed by us; this will permit the analysis of 0.1 ml of serum or 6 mg of protein. A secondary limitation of the thermometric method is the high temperature sensitivity required. The temperature change due to reaction in the titration of 2 g of protein/l. is only 8 m"C. In this work a lock-in-amplifier was used. It has a temperature resolution of 4 p " C (rms). A reasonable alternative is the use of a 100,000 Q thermistor with 2-3 V applied to the bridge. According to a recent paper (49) a noise level of 15 p ° C can be obtained with this apparatus. This is more than adequate for the thermometric titrations described in this work.
ACKNOWLEDGMENT We would like to acknowledge both the Athens General Hospital and the University of Georgia Infirmary for supplying the blood samples used in this study, the support of the National Institutes of Health through Grant Number GM 17913, and the advice and assistance of S. R. Betso. Received for review September 27, 1972. Accepted March 23.1973. M . W. Brown, K . Issa, and A. G . Sinclair, Analyst (London), 94, 234 (1969). D. S. Young, E. K. Harris, and E. Cotlove, Ciin. Chern., 17, 403 (1971), R. J. Henry, Ciin. Chern., 17, 199 (1971). T. Meites, L. Meites, and J. N. Jaitly, J. Phys. Chem., 73, 3801 (1969).
ANALYTICAL CHEMISTRY, VOL. 45. NO. 9 . AUGUST 1973
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