and n-propylbenzene, 0.01 p.p.m. Excellent analytical results have been obtained even at concentration levels four times lower than those shown in Figure 2. For example, in one series of seven experiments in which the initial concentration of m-xylene plus p-xylene was only 0.038 p.p.m., i t was determined that 0.014 =k 0.003 p.p.m. (one U ) were photooxidized to products. Such results are possible only if column substrates sue h as 1,2,3-tris(cyanoethoxy)-
propane, which have good selectivity and excellent stability, are available to use a i t h flame ionization detectors operating a t maximum sensitivities.
(3) McNair, H. M., DeVries, T., ANAL. CHEM.33, 807 (1961); McNair, H. M., Ph.D. Thesis, Purdue University, August 1959; Dissert. Abstr. 20, 2523 (1960); L. C. Card N o . Microfilm 59-6498. C. A. CLEMOXS P. W. LEACH A. P. ALTSHULLER
LITERATURE CITED
(1) .4ltshuller, A. P., Clemons, C. A , , ANAL.CHEM. 3 4 , 466, 747 (1962). (2) Bayer, E. in “Symposium on GasChromatographie-1961,’’ AkademieVerlag Berlin, 1962.
Division of Air Pollution Robert -4. Taft Sanitary Engineering Center Cincinnati 26, Ohio
Determination of Protein Contamination of Deoxyribonucleic Acid by the Fohn-Lowry Method SIR: X simple and dependable spectrophotometric assay for protein contamination would be of value in determining the degree of purity of deoxyribonucleic acid preparations. The FolinLowry test ( 2 , 5 ) , as described by Chou and Goldstein (I), has been used for detection of both protein ( 7 ) and phenol (6) contamination of such preparations. Because of its increasing frequency of use for this purpose, further knowledge concerning the sensitivity of this method and of certain parameters of such a system n-hich might influence color development would be of value in interprcting data obtained in this manner. EXPERIMENTAL
Reagents. Highly polymerized salmon sperm deoxyribonucleic acid (DKA) and a preparation of protamine sulfate were purchased from
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t h e California Corporation for Biochemical Research. Chromatographically homogeneous bases, nucleosides, and nucleotides were purchased from RIann Research Laboratories and Schwarz Bio-Research, Inc. Calf thymus histone was the generous gift of D r . K. Murray of Stanford University. Bases, nucleosides, and nucleotides t o be assayed were dissolved in Folin reagent C. Dilutions 11-ere prepared using this reagent as the diluent. Preparations of protamine, histone, and D N A were dissolved in saline solutions of suitable concentrations. Sodium citrate mas added in 0.01511 concentration to all solutions of DnSA unless otherwise noted. Procedure. A 1-nil. aliquot of the solution t o be analyzed was added, with stirring, t o 5 ml. of Folin reagent C at room temperature. After 30 minutes, 0.5 ml. of diluted (1:l) Folin
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Figure 1 . Color development of certain nucleic acid derivatives with the FolinLowry reagent
1548
ANALYTICAL CHEMISTRY
reagent E was added with vigorous mixing. After 30 minutes, the absorbance of the resulting mixture was determined at 570 mfi. All values so obtained were corrected for coloration of a simultaneous reagent blank. RESULTS A N D DISCUSSION
.in aliquot of salmon sperm D S I containing i 2 pg, of phosphorus per nil. was tested for its ability to produce coloration with the Folin-Lonry reagents. Color development was negligible (ab has been noted ( 1 ) . Deoxyribose derivative- gal e values considerahl> less than those of their ribose analogs, deoxyguanosine and deoxyguanylic acid, a t 0.002X concentrations, giving T alues of 0.090 and 0.029 absorbance units, respectively. Samples of protamine and hiytone were carefully weighed out and disqolved in 1 V sodium chloride. Standard curves relating protein concentrntion to color production nere determined using dilutions of theqe solutions. This experiment was repeated, adding 0.5 ml. of a solution of DK.1 (1 mg. per ml.) in 1,lf sodium chloride to 0.5 ml. of protein solution in 1M sodium chloride. The results are illustrated in Figure 2 in which the curves for protamine and histone, both in the presence and absence of DYA, have been superimposed. While the presence of D N 1 exerts a slight depressing effect upon color
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LITERATURE CITED
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0.050 0.075 0.100 PROTEIN GONG. ( m g / m l ) Figure 2. Color development of protamine and histone with the Folin-Lowry reagent in the presence and absence of DNA
0
prior to analysis. Gel filtration may be used to remove phenol from D Y d (6) isolated by the Kirby aqueous phenol method (41, and this process has been s h o m t o remore bases, nucleosides, nucleotides, and low molecular weight oligonucleotides (5,8). The extreme sensitivity of the FolinLowry assay to the presence of guanine and its ribo- derivatives suggests that it might also be considered as a means for following certain hydrolytic reactions in which these derivatives are released from nucleic acids as reaction products.
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development, this depression is not sufficient to invalidate the quant,itative as1)ectb of this assay. Quantities of protamine and histone c n the order of 10 to 15 pg. per nig. of DKd may be readily detected under these 1:onditions. Dat'a obt,ained during the course of this inre,stigation suggest that the FolinLowry prot'ein assay m a r be used for determining the degree of protein cont,xmiriation during the course of D S - i
isolation and purification, assuming that the color yield of the contaminating protein be known. This situation will be encountered more frequently in the isolation of virus and phage DN.1, each with only one contaminating protein, than in the isolation of DS.1 from more complex sources. The high color yields of xanthine, hypoxanthine, guanine, riboguanine derivatives, and phenol require that these substances be removed
(1) Chou, S. C., Goldstein, -4., Biochetiz. J . 75, 109 (1960). ( 2 ) Folin, O., Ciocalteau, V , J . Bzol Chem. 73, 627 (1927). (3) Gelotte, B., .~atur~zssenschczften48, 554 (1961). ( 4 ) Kirby, K. S.,Bzochenz. J 66, 495 (1957). (5) Lowry, 0. H., Rosebrough, S. J , Farr, -4.L., Randall, R. J J . Bzol. Chem. 193,265 (1951). ( 6 ) Shepherd, G. R., Petersen, 11. F., J Chromatog. 9, 455 (1962). 17) Thomas. C. -4.. Berns. I. I , J . M o l . bioi. 3. 2f7 il961,. (8) Zadrkil, S'.,Sormova, Z., Sorni, F., Collection Czech. Chem. C'ominim. 26, 2643 (1961). GEORGER . SHEPHERD PEGGY A. HOPKISS ~
Los Alamos Scientific Laboratory University of California Los Alamos, N. 19.
WORKperformed under the auspires of the U. S. Atomic Energy Commission.
Conductometric Determination of Alkalinity of Sea Water SIR: The inductii.ely coupled conductivity meter ( 2 ) is now widely used to determine the salinity of sea water. .idvantages of this approach over the conventional AgXOa titration method are its grea, speed, convenience, high accuracy, and small operator error. Furthermore, the instrument is portable, does not require a temperature-controlled bath, and does not have metallic electrodes. Recently n e have applied the inductive conductivity meter to the determination of alkalinif y of sea water. 'l'he major components responsible for the alkalinity of sea water, HC03-. (:03-2, and H2HO3-, inay be converted to H&03 and H3B03 by titration with HC1. The slight chznge in conductivity during the titration is due to the replacement of HCO3-, COa-2, and H2B03- with C1-. fr very sharp rise in conductivity occurs after the equivalence point due to the addition of both C1- and highly conductive H+. The alkalinity of sea water is directly
proportional to the amount of HC1 needed to reach the sharp equivalence point. EXPERIMENTAL
Standard I . O O N HCl is added in 0.050ml. increments, up to a maximum of 0.45 ml., to 10 100-ml. portions of a sea water sample. After mixing, the
Table
I.
relative conductivity ratio of the samples as compared to international standard sea Lvater is measured a t room temperature with an inductive salinity and conductivity meter, Model 621, Hytech Corp.. San Diego, Calif. The reference conductivity of international standard sea water. ohtainable from Standard Sea-TVater Service, Charlot-
Comparison of Two Methods of Alkalinity Determination of Sea Water
Sea water: From the Pacific, 45"h',, 128"W., Februar!. 23, 1963 Temperature of analysis: 23.0" C. Alkalinity, meq./liter _______ Depth, Salinity, pH method Conductivity Difference, Cmeters p.p.t. (1) method /o 0 2.30 32.56 2.3c 0.0 50 2.2s 32.58 2.26 0.9 100 32.96 2.28 0.9 2.30 2.31 2.32 150 33.66 0.4 200 2.33 2.33 33.88 0.0 300 2.34 2.33 0.4 33.98 400 34,04 2.36 2.37 0.4 34.18 2.41 0.4 600 2.40 2.42 800 2.42 34.30 0.0 1000 34.39 2.45 0.4 2.46
VOL. 35, NO. 10, SEPTEMBER 1963
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