triplicate analysis were used for the colorimetric determination of titanium by the hydrogen peroxide method and the remaining one for the colorimetric determination of manganese by the periodate oxidation method. The results of the analysis are presented in Table V. ACKNOWLEDGMENT
The author thanks Ruth Rethemeyer
LITERATURE CITED
(1) Kakihana, H.7 J . Chem. SOC. Japan 72,200 (1951). ( 2 ) Kennedy, J., Wheeler, V. J., -4nal. Chim. Acta 20, 412 (1959).
(6) Ibid:. 32.’1185 (1960’1. (7) Takahafjhi, -T.; Shirai, H., Seisan Kenkyu 10,176 (1958). ( 8 ) Taketatsu, T., J . Cnem. SOC.Japan, Pure Chem. Sect. 79, 586 (1958).
RECEIVED for review October 24, 1960. Accepted December 8, 1960.
Copper Binding by Proteins in Alkaline Solution R.
D. STRICKLAND, M. 1. FREEMAN, and F. T. GURULE
Research Division, Veterans Administration Hospital, Albuquerque,
b The stoichiometry of the biuret reaction of alkaline copper tartrate with proteins, including serum proteins, 6-lactoglobulin, edestin, pork gelatin, gliadin, and zein, is elucidated. In all of these proteins except serum globulin, six peptide nitrogens are bound b y chelation to each copper. In serum globulin, five peptide nitrogens are associated with each copper. The contributions of cysteine, tyrosine, glutamic acid, aspartic acid, histidine, arginine, and lysine to the color of copper protein complexes are discussed. The equilibrium constant for the equimolar complex of histidine with copper as well as its spectral constants are given. Methods for determining peptide nitrogen by means of copper binding and for estimating proteins b y a new titrimetric method are proposed. A method for calculating amounts of proteins measured b y the biuret method, which obviates errors due to erroneous use of reagent blanks, is suggested. Structures of the copper complexes of proteins and of glycylglycylglycine are indicated.
P
in alkaline solution form violet complexes with bivalent copper. Mehl (11) has investigated the amounts of copper bound to human and bovine serum proteins. He suggests that each copper is associated with four peptide nitrogens. Little else has been published concerning the stoichiometry of this reaction, even though it is the basis of the biuret method for estimating proteins colorimetrically. In this method, the color is produced by treating a dissolved protein with a strongly alkaline solution of tartrate-complesed copper. Below p H 10, the principal binding sites in proteins for copper are carboxyl, basic amino, phenolic, and sulfhydryl groups. Of these i t has been possible to saturate only the sulfhydryl groups ROTEIKS
N. M.
which form stable mercaptides. The reaction between copper and the other groups is not quantitative a t any p H ; a t the higher pH’s the products tend to hydrolyze, allowing hydrous copper oxide to precipitate ( 7 ) . KO information has been available concerning the ability of these groups to sequester copper from its tartrate complex, but, because tartrate copper does not hydrolyze in alkaline solution, it seems likely that this ability must be low. If this is true, then virtually all of the copper bound by a protein, under the conditions of the biuret reaction, must be held by chelation to the peptide groups. This would not be true for proteins rich in cysteine. Tshugaev (15) has suggested a structure for the complex between biuret and copper in which two molecules of biuret are bound to a single atom of copper. Freeman et al. (6) have confirmed this structure by x-ray crystallography. This structure should provide a helpful analogy for deducing the nature of the protein compleses, even though the a-carbon groups interspersed between the amido linkages in peptide chains make formation of six-membered rings of the biuret type impossible. The violet color of the complexes is evidence that a t least some of the copprr binding must be by coordination n ith nitrogen; osygen atoms, the only othrr available coordination sites, yield copper complexes with a light blue color. The dependence upon an alkaline medium for the reaction suggests that there may also be binding by peptide groups through enolization. Plekhan and Russianora (12) have demonstrated that racemization of the component amino acids does not occur when copper-protein compleses are subjected to alkaline hydrolysis. Such racemization would be inevitable if the hydrogens from a-carbons were mobilized by enolizittion. Another kind of enolization by which the peptide
group rearranges to form a resonance hybrid of two structures,
O-
(-y=c-I
R
e--* --?;
7)
--c-
has been proposed by Datta and Rabin ( 4 ) to explain the dissociation behavior of copper-dipeptide complexes. Binding by coordination and by covalent linking are complementary, so i t is reasonable to suppose that copper is held to proteins in alkaline solution by both ligand types. Knowledge of the extent of enolization and of the molar ratios of peptide nitrogen to copper in these complexes would go far toward making it possible to write their structures and would provide a theoretical basis for using the biuret method to estimate proteins. This report describes methods by which these objectives have been accomplished. EXPERIMENTAL
Apparatus. All spectrophotometric measurements mere made by means of a Beckman D K spectrophotometer equipped with silica cuvettes having 1-cm. light paths. Measurements of p H were made with a Beckman Model H-2 p H meter, using the E-2 glass electrode recommended for alkaline solutions. A Fisher-Hirschfelder-Taylor kit was used to construct the molecular models. Reagents. Biuret reagent contains 0.02M copper, 0.16M sodium potassium tartrate, and 0.2144 sodium hydroxide. Glutamic acid, aspartic acid, histidine, glycine, arginine, lysine, cysteine, tyrosine, and glycylglycylglycine were obtained as chromatographically pure from M a n n Research Laboratories. p-Lactoglobulin, edestin, pork gelatin, gliadin, and zein were obtained from Nutritional Biochemicals Corp. The biuret was from VOL. 33,
NO. 4,
APRIL 1961
545
Eastman ( S o . 3319), and the urea (biuret-free) was from Mallinckrodt. The inorganic chemicals all conformed to ACS specifications for reagent grade. The serum proteins were prepared from pooled normal human serum by fractionation with half-saturated sodium sulfate according to the method of H o w (8). Procedure. T h e peptide nitrogen in each protein ITas estimated by subtracting amido and basic amino nitrogen from the total nitrogen. Hydrolyzates w r e made by heating t h e proteins with 531' HC1 to 100' C. for 10 hours in sealed glass tubes according to a technique that already has been described (14). The basic amino acids and the ammonia released by hydrolysis were precipitated with phosphotungstic acid ( 2 ) and the nitrogen in the mashed precipitates determined by Kjeldahl microanalysis. Sitrogen measured in this may includes
the 0-amino nitrogen of the basic amino acids nhich must be included with the peptide nitrogen. To make this correction, the basic amino acids were separated from the hydrolyzates by ionophoresis on paper (10)and their amounts estimated by densitometry after staining with ninhydrin ( I ) . Amounts corresponding to the a-amino nitrogen of the amino acids determined in this n a y were then subtracted from the nitrogen found in the phosphotungstic acid precipitates. Total nitrogen was estimated by Kjeldahl analyses of the proteins. All of the nitrogen analyses reported here were calculated from the means of two or more closely agreeing replicate determinations. The method used to estimate the a-amino nitrogen of the basic amino acids is subject t o errors approximating +lo%, but these become small relative to the total peptide nitrogen of a protein. Samples for spectrophotometric study were prepared from fresh stock solutions containing approximately 25 grams of the substances to be investigated per liter. -4number of the proteins did not Table 1. Dilution Schedule Used to yield clear solutions in water, even when Prepare Samples for Spectrophotoelectrolytes were added; thwe (inmetric Measurements dicated in Table 111) were made up in (Biuret reagent contains 0.02 X biuret-free 8 X urea. Comparison exmole of Cut2 per ml. in 0.20M NaOH) periments showed that urea has no demonstratable effect upon the absorpMoles! X 10-3 of Cu+* Grams of tion spectra of protein-copper complexes Grams X 10-8 as Biuret Sample per other than that attributable to the of Sample Reagent Mole of C U + ~ diminution of cloudiness. A seriw of dilutions adjusted to 0.LU 0 0.0 0.100 50 5.0 0,100 in SaOH, containing systematically 100 10.0 0.100 varied proportions of sample and 150 15.0 0.100 copper (as biuret reagent), was prepared 200 20 0 0 100 from each stock solution. Table I 250 25.0 0.100 shows the generalized dilution schedule 313 25 0 0.080 used for all proteins. Samples were 417 25.0 0 060 prepared by buretting appropriate vol625 25.0 0.040 umes of protein standard solutions and 2.5 0 0 020 250 m 25.0 0.000 of the reagent into glass-stoppered test 500 50.0 0.100 tubes. ill1 preparations were adjusted 750 75.0 0.100 to final volumes of 10 ml. with Ka100.0 0.100 000 OH molarities of 0.1 by adding water and 250 125.0 0.100 0 . 2 N KaOH as required. These were 125.0 0.080 563 allowed to stand for 1 hour a t room 0.060 2083 i25.0 temperature, then scanned b e h e e n 0.040 3125 126.0 425 and 850 mp against a 0.1111 Na0.020 6250 125.0 0.000 m 125.0 OH blank. Biuret, glycylglycylglycine, and histidine were subjected t o the
Table II.
Peptide and Terminal Nitrogen in the Substances Investigated
(Terminal nitrogen and peptide nitrogen are not differentiated) % Phosphotungstic Acid Precipiyo Peptide tatable and Terminal Peptide % Total N, Sample Kjeldahl No N by Diff. N* Biuret 40.7 0.0 ... 40.7. Glycylglycylglycine 22.2 0.0 ... 22.2 15.1 1.6 13.5 13.8 @-Lactoglobulin 17.3 4.6 12.7 12.5 Edestin 15.3 2.6 12.7 13.7 Gelatin 16.9 5.2 11.7 11.4 Gliadin 16.1 3.2 12.9 12.5 Zein 15.1 2.1 13.0 12.8 Serum protein 15.1 1.8 13.3 13.0 Serum albumin 15.2 2.8 11.4 12.4 Serum globulin 5
Corrected to exclude a-amino nitrogen of basic amino acids.
* Calculated from published averages (3).
546
ANALYTICAL CHEMISTRY
same treatment using preparations in n hich the sample to copper molar ratios were varied between 0 and 32. The spectral properties of the sample substances alone and of the reagent alone n-ere observed by scanning the first and last dilutions which contained the unmixed substances (Table I). To measure the extent of enolization, 250-mg. portions of protein were dissolved and titrated with 0.3000N Ba(OH)z t o p H 11.5 to neutralize free acidic groups, then a known and excessive amount of Ba(0H)z mas added and the mixture back-titrated to p H 11.5 n i t h 0.200014' CuSO4. At p H levels below 11.5 anomalous results were obtained; below p H 10 the violet color associated with the biuret reaction does not form. Barium hydroxide was chosen for the titration because it gives more consistent results, under the conditions described, than does sodium hydroxide.
RESULTS A N D DISCUSSION
Table I1 shows the nitrogen composition of the proteins. The estimated peptide nitrogen is shown in comparison to amounts calculated from the averages of published amino acid analyses (3). Figure 1 shows the spectral data for complexes of biuret, glycylglycylglycine, and gelatin plotted as molar absorptirity (in terms of copper concentration) against grams of sample per mole of copper, These curves are typical of all of the substances containing peptide linkages that were studied; they will be used to illustrate the methods by which the constants in Table 111 were derived. The absorptivity increases regularly with increasing sample to copper ratio until saturation is attained. Following this point, further increases in the relative sample concentration eauSe a slight, fairly regular decrease or increase in absorbance. This suggests that the availability of excess binding sites may permit the formation of intermediate complexes with atypical light absorptive properties. The curve for biuret breaks sharply with a mixture containing 206 grams of sample per mole of copper; this corresponds to a molar ratio of 2 to 1, the proportion expected from the Tshugaev formula. Absorbance measurements for this curve were made a t 515 mp, the ware length of maximum absorption for preparations containing this ratio of biuret to COPper, so that the absorptivity, 27.8, a t the point where the curve breaks is actually the molar absorptivity, EA-, of the complex. These values differ from the absorption maximum at 505 m p with ehmax = 43 reported by Kober and Haw (9). The values given here are doubtless influenced by the solvent composition, particularly the pH, and
-
= BIURET
80
EXFEi ViiiT4L
A 1 515 m y
FRESIITEZ
CCC = G L Y C Y L G L Y C Y L G L Y C I N E
1/
61 .
AT 5 9 0 m y
:
4 32 05 “
:
2
3
4
5
6
2“
8
32
”
0
200
GRAMS
400
600
800
1000
CF S f i M P - E / M O L E
I200
I400
‘c__ j.
1600
Figure 2. Variations in molar absorptivity of copper in biuret reagent caused b y adding histidine
3 F Cu”
Figure 1. Effects on molar absorptivity of copper in biuret reagent caused b y adding varying proportions of substances that give biuret reaction
Predicted results calculated using dissociation constant for equimolar histidine-copper complex. Measurements m a d e a t 600 mp, the estimated w o v e length o f maximum absorption for this complex
Measurements m o d e a t w a v e lengths of maximum absorotion of complexes
should be taken as valid only for the conditions described in this paper. By the same process of reasoning used for interpreting the biuret curve. i t appears that 1 mole of copper associates n i t h 189 grams i l mole) of glycylglycylglycine to yield a complex with a molar absorptivity of 90.3 a t 590 nip,the r a v e length of its maximum absorption. The point of mavimuni absorbance for gelatin irorrcsponds to a mixture containing GTO grams of the protein for each mole of copper. A t the w a w length of maximum absorption, 554 m p , the molar absorptivity of the complex, calculated on the basis of the copper concentration, is 144.2. The weight of a protein just sufficient to complex n i t h 1 mole of copper, as eytimated in this way, $5.11 be referred to as its “copper-combining n eight” in the ensuing discussion. The substances containing peptide linkngt s that were chosen for this study inc.lude biuret, edcstin, gliadin. glycylglyc~lglycine, &lactoglobulin, zein, gelatin, pooled normal human serum, si’rum albumin, and serum globulin. The spectrophotometric constants found for these substances are summarized in Tablc 111. The nave lengths of ma\imum absorption of the complexes, the molar absorptivitiw of the complexed copper, and the copper-combining neights of the samples were obtained by the method just described. The iiiolcs of pcptidch nitrogen per copper-combining weight were calculated ~ J J using the percentages of peptide nitrogen given in Table 11. Absorbances of copper, as biuret reagent, at the appropriate wave lengths were obtained by measuring dilutions of the
method used to prepare it was deliberately patterned after the clinical method generally used for serum protein fractionations; this is known to yield impure preparations of albumin. It seems likely that the estimations of peptide nitrogen, which are subject to the summation of errors from several analyses, are less accurate than the estimations of copper-combining weight, and that the divergences from integral ratios between peptide nitrogen and copper arise primarily from this cause. If this is true, the deterniination of copper-combining w i g h t offcrs promise as a simple method for finding the peptide nitrogen of a protein. Possible structures of the complexes
reagent. Over the limited range of copper concentrations involved in this work, 0.00 to O.O2M, the biuret reagent follows Beer’s law a t every wave length between 425 and 850 mp. The significance of the eopper-conibining weights of biuret and of glycylglycylglycine already have been discussed. Table I11 shons that all of the proteins except serum globulin have combining neights that contain siu pcptide linkages if the true numbers are taken t o be the nhole numhcr nearest to those calculated. The lo^ value found for serum albumin reflects its admivture n ith a-globulins. The
Table Ill.
Constants Relating to Spectrophotometric Measurements of Substance Giving Biuret Reaction Molar Absorptivity a t Moles of
2.8
Cu-Combining Wt., mc 206
Peptide/ Cu-Combining Rt. 5.99
90.3
13.9
189
3.00
545 550
151.4 163.6
6.2 7.6
620 635
5 98
(-75,000)
554
144.2
8.0
670
6.27
( 27.000)
555
143.4
8.3
700
5.92
535 545
150.0 163.3
5,4 6.2
638 618
5.83 5.39
545
165.9
6.2
632
5.55
545
154.0
6.2
586
4.94
Xrnax
Sample and M.01. iVka Biuret (103) Glycylglycylglycine 1189) (189) p-Lactoglobulinb p-Lactbglobulinb (41,600) Edestinb (310,000)‘ Gelatin, pork
CuX of Complex 515
Of complexed Cu 27.8
590
A,,
Gliadinb
Ze&’ ’ (45,000) Total serum protein Serum albumin (70,000) Serum globulin (180 000)
CUX Of biuret reagent Cu
0
Taken from Spector (IS).
c
Weight in grams just sufficient to complex 1 mole of copper.
6,72
* Dissolved in 8M urea.
VOL. 33, NO. 4, APRIL 1961
547
formed by proteins (I) and by glycylglycylglycine (11) are shown belon-.
cu
I
>,C
1
\o/c\N
N//c\o/
/
H,C-NH,
HCR
H
'c-6
0 . t '
/I
HN
a,
HCC
H
0-c
HN
'NH
2 . O
I1
,,k ...c u h C R N//C\@/ \o/c\N :'
I
I
/
\
I A simple molecular model that accounts for the ratio of six peptide nitrogens to each copper atom and allows for bonding by enolization as well as by coordination can be made b y following Structure I. The two peptide groups in this structure that do not participate in copper binding may be enolized or not, depending upon the specific protein. The peptide chains in I are arranged as parallel helices while that of I1 is folded. The ligands to the copper atoms in both structures are in square planar configuration. I n Structure I each copper is coordinated with a nitrogen from either chain and is combined covalently with the succeeding pair of enolized peptide groups. The next pair of peptide groups is too widely separated to permit mutual participation in the binding of copper. This separation is very evident in an actual model; a similar situation has been reported with respect to the two central groups in the copper-biuret complex where i t was observed by means of x-ray crystallography (6). I n a molecular model of the indicated structure, the ligands to each copper atom fall into a square planar configuration while the peptide chains automatically become arranged into mirror-image helices having the same atomic distribution as the familiar a-helix. The insertion of enolized peptide linkagesinto the chains at the points indicated does nothing to disturb the helical form. This structure is suggested only as a hypothesis; i t serves to explain the composition of the complexes and it will be useful for explaining the results obtained by titration. KO claim is made for its validity. It is tempting to speculate that the complex formed with serum globulin niay have an entirely different structure involving only five nitrogens for each atom of copper and to suppose that the difference arises from the geometry of the protein molecule. A model of an intramolecular complex in which every fifth nitrogen is excluded from participation in copper binding by its geometric position relative to the rest of the chain can be constructed. I n such
548
0
ANALYTICAL CHEMISTRY
a model the copper ligands fall into square planar configuration when the coordinating nitrogens are adjacent with their attached hydrogens turned outward. The differences in absorptivities and in copper-combining weights among the copper complexes of different proteins make the estimation of one kind of protein by comparison to standards prepared from another poor analytical practice (Table 111). It is fortunate that the clinical measurements of serum protein, which frequently are based on albumin standards, are not seriously affected by these differences; the lower absorptivity of the copper-globulin complex is nearly compensated for by its lower copper-combining weight so that the error inherent in assuming that the two fractions do not differ spectrophotometrically causes the globulin to be estimated only 2.2% high when albumin is taken as the standard substance. The spectrophotometry of copperprotein complexes in the presence of excess biuret reagent is complicated b y the fact that the unbound copper displays appreciable absorption over the wave length range best suited to estimating proteins. If copper is assumed to be present in two forms, free and as the complex, the treatment of absorption data can be simplified by algebraic manipulation. I n this case the total absorption of a sample will be
+
At = A C ~ P A ~ u
(1)
where AcUp is the absorption attributable to the complex and Ac,, that caused by unbound copper. By using the molar absorptivities of the complex and of free copper, taken at the wave length of maximum absorption of the complex, substitutions can be made to obtain At = ecup[CuPI
+
[CUI
(2)
I n the biuret method for estimating proteins, a fixed amount of the reagent is added t o a fixed aliquot of protein solution. I n most procedures the biuret reagent is 0.02M in copper and is added volume for volume to the sample to yield a preparation that is 0.01M in copper. Using this concentration as a n example and allowing for the distribution between bound and free copper, Equation 2 can be written as (0.01 - [CUP]) At = €cup [CUP]
+
(3 ) or
At
=
(€cup - Ecu) [CUP]
+ 0.01 E ~ u
At any given wave length the difference between the molar absorptivities of a copper-protein complex and of unbound copper is a constant, eCuP - eC,, = 4 e , and the quantity 0.01 eCu is also a
constant which will be designated as D. Substituting 4~ and D into Equation 3 and solving for the molar concentration of coniplexed copper gives [CUP]
=
-D A€ At
(4)
When both sides of this equation are multiplied by the copper combining weight, m,
is obtained. This can be written as
P
m - (A, - D) Ae
(6)
since m [CUP] = P , the concentration of protein, in grams per liter of the preparation being subjected to spectrophotometry. The concentration calculated in this way, multiplied by a factor to account for dilution, gives the concentration of protein in a sample solution. It also obviates the error inherent in reading sample preparations directly against a reagent blank, a practice involving the tacit assumption that the concentration of free copper remains constant in the presence of any amount of protein. The values of m for various proteins have been given in Table 111, those for 4e,D, and m/Ae are shown in Table IV. iipplying this equation to preparations containing known amounts of protein tests the accuracy of the biuret method. For this purpose, appropriate amounts of the stock solutions were measured into test tubes, diluted to 5.00 ml. with water, and mixed with 5 00-ml. portions of the biuret reagent. All volumes were measured with ordinary 50-ml. burets. Each substance was measured a t the wave length of its maximum absorption (Table 111). The results obtained are summarized in Table V. The apparent differences in accuracy of estimation among the various substances are not statistically significant. Comparison of the variances yields F = 0.177; since the critical value for F (1% level) is 2.11, it was concluded that all substances had been measured n i t h uniform accuracy. Pooling all results gives a mean recovery of 99.73 i- 2.22(S.D,)Y0over the concentration range 0.5 to 3.0 grams per liter of protein in the preparations being subjected t o spectrophotometry. This allows errors in determining individual samples not to exceed +6.00Y0 within 99% confidence limits. The molecular weights of the proteins given in Table I1 were taken from Spector ( I S ) . There is no correlation between the wave lengths of absorption maxima and the molecular weights. This is in contradiction to the statement made by many textbooks that proteins of low molecular weight yield red complexes Lvhile those with high
molecular weights yield blue complexes. There are visible differences in the colors, but these appear to be unrelated to molecular weight. KO direct approach is available for studying the effects of the amino acid side chains upon the absorption spectra of copper-protein complexes, but a semiquantitative estimate of these effects can be obtained by examining preparations containing biuret reagent and the free amino acids. When cysteine was mixed w-ith the reagent, an immediate and quantitative reaction, yielding the familiar olivecolored cuprous mercaptide, occurred. The tendency toward this reaction is so marked that even copper already bound to a cysteine-free protein such as gelatin can be sequestered quantitatively by adding stoichiometric amounts of cysteine. The same reaction occurs with cystine, but a t a slower rate, as would be expected from the fact that hydrolysis of the disulfide linkage is a necessary preliminary to mercaptide formation. The absorptivity of the mercaptide is large; it increases steadily with decreasing wave length between 850 and 425 mp. I t displays erratic spectrophotometric behavior, particularly with respect to dilution, so that absorption constants have not been calculated. Evidently proteins rich in cysteine cannot be estimated accurately by the unmodified biuret method. I t is surprising that proteins-for example, serum albumin-that contain large amounts of cystine do not show this effect since the disulfide cross linkages might be expected to hydrolyze under the conditions of the reaction. Lactalbumin, the albumin-V fraction of Cohn, and fibrinogen do react to form mercaptides. This is unfortunate, particularly in the case of fibrinogen, which often is estimated, for clinical reasons, by means of the biuret method. It is doubtful that the results of such measurements have much quantitative meaning. Fibrinogen was to have been included in this study, but the spectrophotometric results obtained cyere so aberrant that the experiment was abandoned. The other amino acids with copperbinding side chains have effects of smaller magnitude. When tyrosine, lysine, arginine, glutamic acid, or aspartic acid are added to biuret reagent, even in amounts equimolar with the copper, the effects upon the absorption spectrum are negligible. This is true whether or not a protein is present in the miuture. It does not matter, for the purposes of this report, whether this failure of the reagent to change color results from similarity between the spectra of the amino acid complexes and that of tartrate-copper or from inability of the complexes to form in competition with tartrate. The latter
is probably the more important factor since additions of large excesses of any of the above acids do cause changes in the spectrum of the reagent Whatever the reason, these acids have no special role in forming the biuret color. Biuret reagents prepared by substituting any of the above amino acids for tartrate in the formulation are still able to give good color when mixed with protein; this indicates that the individual amino acid complexes are much less stable than those formed by peptide chains. Some idea of the great stability of copper-protein complexes can be had by noting that even (ethylenedinitri1o)tetraacetic acid (EDTA) can be used as the complexing agent in preparing the biuret reagent; here, however, there is a noticeable reduction in intensity of the color produced by adding protein.
Table V.
Compound Biuret
Glycylglycylglycine
8-Lactoglobulin
Edestin
Gelatin
Gliadin
Zein
Serum protein
Serum albumin
Serum globulin
Table IV. Constants Used to CaIculate Concentration of Substances from Absorbance Measurements of Complexes Formed in Biuret Reagent
(These constants are to be used in Equation 4) Substance Serum albumin Serum globulin Serum total protein @-Lactoglobulin Edestin Gelatin (pork) Gliadin Zein Biuret Glycylglyc~lglycine
m/Ae
Ae
L)
3.957 159.7 0.062 3.965 147.8 0.062 3.934 4,270 4.071 4.919 5.181 4.412 8.240 2.474
157.1 145.2 156.0 136.2 135.1 144.6 25.0
0.062 0.062 0.076 0.080 0.083 0.055 0.028
76.4 0 139
Accuracy of Measurements by the Biuret Method
Grams per In sample 0.21 0.41 0.62 0.83 1.03 0.36 0.72 1.06 1.42 1.78 0.52 1.04 1.55 2.07 2.59 0.52 1.04 1.56 2.08 2.60 0.50 0.99 1.49 1.97 2.47 0.51 1.02 1.53 2.04 2.55 0.52 1.04 1.57 2.09 2.61 0.56 1.14 1.70 2.28 3.84 0.59 1.18 1.77 2.36 2.95 0.60 1.20
1.80 2.40 3.00
Liter Found 0.21 0.43 0.61 0.82 0.99 0.35 0.72 1.06 1.45 1.79 0.53 1.06
1.54 2.02 2.49 0.53 1.04 1.57 2.04 2.52 0.48 0.99 1.51 1.99 2.51 0.51 1.04 1.54 2.06 2.48 0.53 1.06 1.57 2.06 2.55 0.56 1.14 1.71 2.27 2.81 0.58 1.21 1.77 2.35 2.76 0.61
% ’ Recovery 100.0 104.9 98.4 98.8 96.1 97.2 100.0 100.0 102.1 100.6 101.9 101.9 99.4 97.6 96.1 101.9 100.0 100.6 98.1 96.9 96.0 100.0 101.3 101.0 101.6 100.0 102.0 100.7 101.0 97.3 101.9 101.9 100.0 98.6 97.7 100.0 100.0 100.6 99.6 98.9 98.3 102.5 100.0 99.6 93.6
Mean Recovery With Std. Dev., %
99.62 f 3.24
99.98 i 1.80
99.38 f 2.60
99.50 5 2.00
99.98 f 2.29
100.20 f 1.80
100.02 j=1.87
99.82 f 0.64
98.80 f 3.28
1.18
1.82 2.40 2.97
VOL. 33, NO.
4, APRIL 1961
549
Histidine requires special attention because it produces a strong color with biuret reagent’. When preparations with inolar ratios of histidine to copper ranging between 0 and 32 were examined spectrophotometrically and the results plotted as molar absorptivities, calculated on the basis of copper concentration, agninst the molar ratios of the rc>actants, the curve obtained ( r3 ’1 gure 2) differ,d greatly from the corresponding curve for substances containing peptide linkages (Figure 1). Instead of straight line relationships with a sharp break a t the point representing complete binding of the copper, the plot falls into :i number of distinctly different stiges. Within the interval including histitline to coppc’r ratios bclow 1.5, thr c-urve bends smoothly in the manner to be cspected for an (,quilibiium reaction n-hen the conc.xtration of one reactant is increased. Betwecn the molar ratios of 2 and 6, the absorptivit’y increases in a linear way as if the additional histidine were adding quantitatiwly to the hist’idinc-copper complex formed in the previous range. Varying the molar ratios between 6 and 16 causes no change in absorptivity, but rat’ios beyond 16, to 32 display slight, apparently linear, increases in absorptivit’g. The entire curve is shon-n here as a m ittcr of general interest. Specific inter st can be confined to the first segment 0:’ the curve. This limitation is made possible by the fact that the separation of histidine side groups by 0thF.r components of a protein chain make it unlikely that more than one group \vi11 be available to a single copper atom; pragmatically, no protein, in the amounts used for the biuret test, is likely to contain enough histidine to supply imidazole groups in concentrations cxceeding that of the free copper. lkasuremcnt of the molar absorptivity. and hence of the concentration, of the 1 to 1 histidine complex in different mixtures of the reagents requires a different approach than that used for the protein complexes. Here secondary complexes begin to form before the initial reaction is complete. It is possible, by using algebraic method?, t o estimate the molar absorptivity of the equimolar complex by extrapolation. For this purpose the equation for the reaction, as a dissociat’ion is w i t t e n in its simp!est form, CuH
e Cu
+H
where CuH represents the complex, C u the copper in unchanged biuret reagent, and H the free histidine; tartrate can be neglected because there is a sufficiently large excess in the reagent so that its concentration will change little, whether or not that held by copper is liberated by the reacting histidine. The dissociation constant for a reaction
550
ANALYTICAL CHEMISTRY
is invariant so i t is permissible to write
-[Cu] - ’[HI
I
[CuH]I
[CUI”[HI’I
(7)
- [CuH]”
when the prime marks represent different concentrations of the reactants in two equilibrium mixtures. When the total concentrations of copper and histidin? are known, the concentrations remaining in the free form n hen equilibrium has been reached can be represented by ([CUI, - [CuH]) and ([HI, - [CuH]) if the subscript zeros denote the initial concentrations of the added reactants. The relationship then becomcq ([CUI,’ - [CUH]’)([Hlo’ - [CuH]‘) [CuH]‘ ([C‘U~O‘’ -~ ~ _[CuH]“) _ ([Hlo’ - [CuH]”) [CuH]”
be evaluated. At 650 mp, a wave length at which both reagent and complex absorb strongly, E = 01.3 for the complex and 25.3 for the reagent. With AEknown, the concentration of the equimolar complex of histidine in expcrimental preparations can be calculated by using Equation 4. This information can in turn be used to compute the dissociation constant, which n as Casting found to be 12.23 X the dissociation relationship into its quadratic form
+ [HI0 + + [Culo[Hlo
[CUH]’- ([CUI, [CuHl
+
[CUI”’) ([CuH]’)’} [Cu]o”[H]o”[CuH]”([H]o” [ C U ] ~ ” ) (ICUH]”)’(9)
+
+
The only unknown quantiticc in this equality are the conccntraiiom of CuH. These can be replaced by
At - D A€
from the same reasoning used
to arrive a t Equation 4. This J ields
=
0
(13)
enables the calculation of [CuH] for preparations where [Cul0 and [HIoare known. If these calculated values are used with Equation 4 to predict absorptivities
(8)
which can be expanded t o
k~ias)
At
=
AE[CUH]- D
(14)
and these are compared to those found for experimental preparations, the applicability of the dissociation concept is tested. This comparison has been made in Figure 2 where agreements up to the 1.5 to 1 histidine-copper ratio are excellent. K i t h the proportions of histidine-copper and Erce copper calculable, i t is a simple matter to construct the absorption spectrum for the complex by subtracting the absorption attributable to free copper at each
[-y]” + + ([*e]
[ C U ] ~ ” [ H]~”
([Hlo”
When absorption measurements of the two equilibrium mixtures are made, [&I$?]”
[!52]
can be evaluated because
the AE’S cancel. Let this quotient be r. Nultiply through the equation by (A€)*. This results in ~[Cu]o’[H]o’(Ae)’ - ?(Ai - D)”X ([Hlo’ [CUIO’)AE r [ ( A t - D)”]’ = [ C U ] O ” [ H ] O ‘ (-A ~(Ai ) ~ - D) X ([Hlo” [CU]O”)AE 4- [ ( A i - D)”]’ (11)
+
+
+
Transposing all terms to the left and collecting like quantities yields a(Ae)’
- bAe
+c
=
0
(12)
where a, b. and c represent the values found by collecting the known terms. This equation is in standard quadratic form and can be solved for AE in the usual way. The quantity Ae has already been defined as the difference between the molar absorptivity of a complex and that of the copper in biuret reagent a t the same wave length; since eCu is easily obtained, eCuH can
[CU]~”)
(10)
wave length from the spectrum obtained with a preparation containirg the complex. This has been done over the 500 to 700 mp wave length range; the result is shown in Figure 3. At 600 mp, the wave length of maximum = 109.7, and at 545 absorption, E,,, mp, E = 83.3. Such strong absorptivity indicates that histidine cannot be neglected as a chromogenic source in the biuret reaction of proteins. It can be calculated by means of the equations and constants given above, that a hypothetical protein containing 2.5% histidine, with m = 650 and emax (545 mp) = 150, would have an absorbance, in 1-em. cuvettes, of 0.780 if the preparation of i t being subjected to spectrophotometry contained 3.25 grams per liter. Of the total absorption 0.012 or 1.5% vvould be attributable to histidine. It is possible that this effect may be either greater or less with histidine incorporated into the protein molecule than i t is with free histidine; in either event further study is indicated. Once the acidic groups of the protein
' I10 20
t b-
!O 9O 0
I
Figure 3. Calculated absorption spectrum of equimolar histidine-copper complex
1 '
70 500
520
540
560
5@0 n4vE
600
620
640
660
6 @ C 700
720
LiNGTd, -p
are neutralized and the p H has been raised to 115, a pH a t which the biuret reaction occurs freely, the titration of a protein with barium hydroxide and copper sulfate TTould be expected to follow a course indicated by the equation
The fact that proteins vary in their abilities to neutralize hydroxide in excess of the amounts required for copper binding and the fact t h a t the ratios of peptide nitrogen to excess hydroxide are not integrals as would be expected if the enolization occurred -O--Cu-O-(!
I1
+ 2Hz0 + BaSOl
I This equation docs not differ stoichiometricnlly from that to be w i t t e n for the two reagents in the absence of protein so that the only indication for enolization of peptide groups would be in the failure of copper hydroxide to prccipitate. ilctually the reaction docs not have the expected stoichiometry. Instcad, quantities of alkali greatly in excess of those to be accounted for by reaction in conjunction with copprr are utilized. The results of such titrations are shown in Table VI. Undcr the specified conditions the amount of hydroxide varies among different proteins but is constant for any given protein. From Structure I, enolization of peptide linkages beyond those required for the binding of copper can be accounted for by assuming the enolization of those groups that are prevented from participating in the complex by their geometric positions Such a hypothesis would permit up to 33y0 of the peptide nitrogens to act as acidic sites supernumerary to the copper requirements; this is adequate since only about 207, are found in actual titrations. If globulin really forms intramolecular complexes as has been suggested, the percentage of supernumerary sites found (22.97,) is slightly higher than is permissible on the assumption that only one peptide nitrogen in five is available for enolization of this kind; it is possible t h a t this anomaly may arise as the result of the same effect as will be described for glycylglycylglycine.
in a quantitative manner, remain to be explained. It seems likely that peptide groups may vary in enolizability by virtue of the influence of adjacent side chains and that the enolization itself is a n equilibrium reaction R hen copper cannot act as a stabilizing agent. The chemistry of the concurrent titration of proteins with alkali and copper h3s proved to be involved and is still being investigated; the subject has been introduced a t this time only to support the proposition that enolization of the peptide linkages does occur. Biuret, with two supernumerary acidic sites, behaves titrimetrically in the way to be expected from the Tshugaeff formula, but glycyIglycylglycine titrations take a surprising course that givrs evidence for a mechanism in addition to enolization by TT hich hydroxides can react with a copper-peptide complex. From the 1 to 1 molar ratio of glycylglycylglycine to copper that has been established by spectrophotometry, Structure I1 is an obvious deduction. This structure seems to require t h a t tv-o and only tn.0 equivalents of alkali per mole n-ould be consumed in its formation, but the fact is that an equimolar mixture of copper and glycylglycylglycine can consume exactly four equivalents of alkali. This titration goes smoothly and quantitatively. Because of its peculiarity i t has been verified again and again using varying amounts of the tripeptide, Titration with sodium instead of barium
hydroxide does not affect the results. It seems inescapable that this complex, in solution, at p H 11.5, yields two protons by the ionization of hydrogens attached t o a-carbons in the glycine residues, Dobbie (6) has described the utilization of three equivalents of alkali by this peptide during the forniation of a n equimolar complex a t pH 10.9, but the utilization of a fourth cquivalent in the pH range 11 to 11.5 has not previously been reported. The structure for the copper-pcptide complex indicated here does not conflict with the tridentate formulation, involving 2 moles of tripeptide for each mole of copper. that Dobbie has proposed. Such a structure seems possible n hen an excess of peptide is available; the structure shown here would be expected to form n i t h excessive or equimolar amounts of copper. The fact that reproducible titrations of proteins with barium hydroxide and copper sulfate can be obtained under the conditions described suggests the possibility of a new method for eqtimating proteins. The rcsults of an experiment to determine the reproducibility of the titration, using pork gelatin as a sample,
Table VI. Supernumerary Acidic Sites in Substances Containing Peptide Linkages
Equivalents per Cu-Combining w t . of: Super- Peptide numer- and terary minal A as a acidic nitrogen, Percentsites, A B age of B
Sample Biuret 2 00 Glycylgl~cJ~l2.00 glycine ,&Lacto1.17 globulin 1.12 Edestin 1.27 Gelatin 1.24 Gliadin 1.19 Zein Total serum protein 1.36 Serum albumin 1 . 4 3 Serum globulin 1 . 1 3
6 00
33.3
3.00
66.7
5,98 5.72 6.27 5.92 5 83
19.6 19.6 20.3 20.9 20.4
5.39 5.55 4.04
25.2 2i.8 22.9
Table VII. Precision for Titrating the Supernumerary Acidic Sites in Pork Gelatin Equivalents of
Sample 1
2 3 I
5 6 Mean Std. dev.
Ba(0H)Z Consumed upernumerary in a Gram of Gelatin, x
3:
1 ,894 1.920 1.899 1.876 1,846 1.887 1.887 & O , 025
VOL. 33, NO. 4, APRIL 1961
551
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 equivalent was added 18.000 X 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.
