Evaluation of Three Methods of Protein Analysis for Serum and Heart Homogenates
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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 2 Copyright 1979, Institute for Clinical Science, Inc. Evaluation of Three Methods of Protein Analysis for Serum and Heart Homogenates JON ATH AN E. SCHWARTZ, B.S.* and BEATRICE C. DURHAM, M.A. William Likoff Cardiovascular Institute, Hahnemann Medical College and Hospital, Philadelphia, PA ABSTRACT The Biuret, Ultraviolet and Lowry techniques for the determ ination of total protein content were compared in 31 samples of patient serum and in 30 rat heart tissue homogenates. Statistically significant differences were found to exist betw een values obtained with each method. Human serum and heart homogenate protein were both lowest w hen assayed by the Lowry procedure, while serum and homogenate values were highest w ith the Ultraviolet and Biuret method, respectively. Possible sources of these discrepancies are discussed. Although the Biuret m ethod may not be as sensitive as the other two methods, it proved to be the m ost reproducible technique for determ ining total protein concentration in the particular types of samples used in this study. Introduction Studies utilizing tissue enzym es to measure rates of metabolic activity must have some means of normalizing values so that various samples can be compared. The most direct m ethod is to utilize wet w eight of the tissue.1 Dry w eight is another m eans o f relating enzym atic activity to the tissue sam p le.14 H ow ever, because the active portion of the tissue may differ from organ to organ, weight may not be a very accurate reference value, and protein content is often preferable. N itrogen determ inations have been em ployed for the quantitation of tissue p ro te in s,3 b u t since th ese are tim e consuming, many investigators now use one of the various spectrophotom etric m ethods for assaying protein. Peters reported the relative popularity of a variety o f sp e ctro p h o to m etric m ethods and showed that some form of the biuret reaction was being used by the majority of laboratories surveyed.15 All soluble proteins give the biuret reaction w hich occurs upon the treatm ent of a peptide or protein with C us 04 and alkali.20 The resulting purple complex of Cu2+ can be q u a n tita tiv e ly m easu red by sp e c tro photometry.9 The m ethod of Gornall et al6 was som ew hat p referred over that of W eichselbaum 20 in a 1964 survey.15 Other laboratories use a m odification of the F o lin -C io c a lte u 2 or Low ry p h e n o l * Present address: Temple University School of technique10 which combines the biuret Medicine, Philadelphia, PA reaction w ith the reduction of the Folin /79/ $00.90 Institute for Clinical Science, Inc.
2 140 SCHW A RTZ AND DURHAM phenol reagent by the copper-treated protein, relative to its tyrosine and try p tophan content. U ltraviolet (UV) spectrophotom etric methods are also used in the quantitative estimation of protein concentrations.8,16,17 Proteins exhibit an absorption maximum in the region o f280 nm. This absorption is caused by the aromatic amino acids present in protein, specifically tyrosine and tryptophan. The intensity of absorption at this wave length depends on the proportions of these amino acids in the protein. However, the base components of nucleic acids or nucleotides, purine and pyrim i dine, also absorb strongly at 280 nm. Fortunately, nucleic acids absorb light more strongly at 260 nm than does protein. W arburg and Christian capitalized on this fact and devised a series of calculations which are m eant to elim inate the interference ow ing to nucleic acids.18 Lowry is the most sensitive of the three methods, capable of m easuring as little as 200 ng protein per sample. Its sensitivity is 10 to 20 tim es that of the UV determ ination and 100 tim es more than biuret.10 Parvin et al have reported that the Lowry m ethod is preferable to the b iu re t reaction for determ ining protein in crude tissue extracts13 and Gonyea et al5 have determ ined that the Lowry m ethod is most appropriate for estim ating protein in isolated ferritin preparations. On the other hand, Lubran recom m ended the biuret m ethod for the determ ination of total serum protein.11 Since both the nature of the sample and the level of protein concentration to be determ ined can affect the resu lts, th e b iu re t, Low ry and UV techniques were compared by us on the same samples of both human serum and rat heart disease hom ogenates. Materials and Methods Patient serum was obtained from 31 patients adm itted to the Coronary Care Unit at Hahnem ann Medical College and Hospital. T hree tissue hom ogenates w ere prepared from each heart often male Wistar rats, 200 to 400 g. The rats were sacrificed by a sharp blow on the head, following which the heart was immediately excised and placed in cold saline. Pieces of heart tissue w ere w eighed, m inced w ith scissors and h o m ogenized by a motor-driven teflon pestle in m edium (25 ml per g) containing 0.25 M sucrose, M m ercaptoethanol and M n eu tralized sodium ethylene-diam inetetraacetic acid. T h e hom ogenate was centrifuged at 16,000 g for 10 min at 4. The supernatant fraction was rem oved and centrifuged for an additional 10 min. Standard curves for the Lowry and b iu re t m ethods w ere p re p a re d from bovine serum album in (Fraction V); control serum and protein standard solution were assayed in order to assure continued accuracy. The biuret protein assay was perform ed according to the m ethod described by Gornall et al.6 Protein estim a tion by UV absorption was carried out according to the m ethod of W arburg and Christian18 as outlined by Layne.8 The Foliri phenol technique was followed according to the m ethod of Lowry et al.10 All samples were analyzed in duplicate by each method. Patient sera were analyzed first by the UV method, then by the biuret and Low ry procedures. T issue hom ogenates were analyzed by the biuret, then by UV and Lowry methods, except for six samples on which biuret values were obtained following the other two values. W hen samples were not processed on the same day by all three methods, they were stored frozen. Results The mean estimates ± standard error of the mean of total protein for each m ethod are given in tables I and II. Human serum assayed by the biuret method had a ± 0.73 mg protein per ml, while the mean values obtained with UV and Lowry were
3 PRO TEIN ANALYSIS O F SERUM AND HEART ± 2.06 and ± 0.83, respectively. One-way analysis of variance b e tw een the values obtained on serum using the three m ethods was significant at p < Using a Student s t-test for related samples, the mean differences betw een biuret and Lowry and betw een UV and Low ry w ere significant at p < Biuret was found to be significantly lower than UV (p < 0.01). Biuret determ ination of rat heart homog en ates (table II) y ield e d a m ean of 1.20 ± 0.07 mg protein per ml while UV and Lowry were 0.82 ± 0.04 and 0.34 ± 0.004, respectively. M ean differences b e tw een thte values obtained from homogenates by the three m ethods and one-way analysis of variance of the three groups of values w ere all significant at p < W ith both types of samples, Biuret and UV values w ere higher than those obtained with the Lowry m ethod (p < 0.001). H ow ever, b iu ret yielded considerably h ig h er values w ith hom ogenates and somewhat lower values with serum than did the UV method. Discussion The results stress the im portance of considering the relative advantages and limitations of any m ethod used to analyze protein concentrations, as w ell as the nature of the samples to be assayed. Significantly different mean values for total protein of both tissue and serum were obtained with each of the three methods. How ever, the differences betw een the UV and biuret m ethod for hum an serum may be within the limits of the determ ination since the difference represents only 8 percent of the value. This was not the case with the heart homogenates, where the differences w ere 30 percent. Results obtained by the Lowry m ethod w ere consistently lower than those obtained by either of the other methods. In addition, control serum values (acceptable range 29 to 32 mg per ml) w ere lower using the TABLE I T otal P rotein in Human Serum M ean Mean D i f f e r e n c e D i f f e r e n c e Mean ± S.E. fro m UV fro m Low ry M e th o d m g /m l m g /m l m g /m l Biuret ± ± ± 1.1( p<0.01 Lowry ± ± 2.06 UV ± 2.06 n = 31 Lowry m ethod (26 mg per ml) but were 32 and 33 mg per ml for the UV and biuret methods, respectively. The coefficient of variation for duplicate determ inations was 0.95 percent for biuret, 5.1 percent for Lowry and 13.4 percent for the UV method. Various drawbacks which have been described in the literature could partially account for our disparate results.6,7,10,15,21 M any substances have been shown to interfere w ith color developm ent in the Lowry method,4 including sucrose which was present in our homogenates. While ethylene diam inetetraacetic acid (EDTA) is known to reduce the Folin-Ciocalteu phenol reagent,12 its presence in the samples would only have tended to increase the values, not to decrease them as we observed. Difficulty was encountered by us in preparing the cupric sulfate-sodium potassium tartrate rea g e n t such as re ported by Zishka and Nishimura.21 W hen TABLE I I T otal Protein in Rat Heart Homogenates M ean Mean D i f f e r e n c e D i f f e r e n c e M ean ± S. E. f r o m UV f r o m L o w ry M e th o d m g/m l m g /m l m g /m l Biuret 1.20 ± ± ± 0.06 Lowry 0.34 ± ± 0.03 UV 0.82 ± 0.04 n = 30
4 142 SCHW ARTZ AND DURHAM the present authors assayed control serum using their procedure, higher values were obtained than w hen using reagent prepared according to Lowry. Although the UV m ethod produced results w hich w ere w ithin a reasonable range, certain disadvantages becam e ev i d en t as our study progressed. The relatively high dilutions necessary to read the values in a suitable scale on the spectrophotom eter may have contributed in part to the greater variation of values seen with hum an serum. Also, since album in contains comparatively few aromatic amino acids responsible for UV absorption, the determ ination of total serum protein by this m ethod has been discouraged when the album in concentration is unknown.19 While W arburg and Christian attem pted to m inimize interference due to nucleic acids, the higher values obtained with serum and the lower ones w ith homogenates may be indicative of im proper correction for their presence in the two types of samples. In addition, simple peptides exhibit maximum absorption at 190 nm rather than at 280 nm.19 Their presence or absence would affect the biuret more than the UV values. O f the m ethods used in this study, biuret has yielded consistent results with relative speed and ease. It does not require the high dilution necessary for the UV determ ination and nucleic acids do not interfere. Although biuret is not as sensitive as Lowry,10 the color yield per weight of peptide is more constant from protein to protein,2 and the presence of sucrose or EDTA does not affect the reaction. W hile the final choice of m ethod rests w ith the investigator and the special needs of a particular study, it w ould appear that the biuret m ethod has more versatility and accuracy w hen dealing with a variety of samples with different components, in which the quality of protein available for assay is not a lim iting factor. A cknowledgm ents This work was supported in part by P.H.S. Grant 5-T ol-h L H T A during the tenure of Jonathan E. Schwartz as a Pre-Doctoral Fellow. The authors w ould like to thank Dr. Victor Satinsky whose perservance and enthusiasm made the Pre-Doctoral Program and this project possible. References 1. Belliv e a u, R. E. and COVINO, B. G.: Cardiac metabolism during hypothermia. Arch. Int. Pharmacodyn. 280: , F olin, O. and ClOCALTEU, V.: On tyrosine and tryptophan determ inations in proteins. J. Biol. Chem. 73: , FRANKEL, S.: Gradwohl s Clinical Laboratory Methods and Diagnosis. 7th ed., Vol. 1, Chapter 7, Nitrogen, pp , C. V. Mosby, St. Louis, G er h a r d t, B. and Be e v e r s, H.: Influence of sucrose on protein determination by the Lowry procedure. Anal. Biochem. 24: , G o n y e a, L. M., L a m b, C. M., Su n d b e r g, R. D., and D ein a r d, A. S.: Comparison of three procedures for isolating human ferritin, for use as a standard in an immunoradiometric assay. Clin. Chem. 22: , G o r n a l l, A. G., Ba r d a w il l, C. J., and D avid, M. M.: Determination ofserum proteins by means of the Biuret reaction. J. Biol. Chem. 177: , G roves, W. E., D avis, F. C., and Se l l s, B. H.: Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Anal. Biochem. 22: , Layne, E.: Spectrophotometric and Turbidim etric M ethods for M easuring Proteins. Methods in Enzymology, Vol. Ill, pp , Le h n in g e r, A. L.: Biochemistry. Worth Publisher, New York, 1975, p Low ry, O. H., Ro sebr o u g h, N. J., F arr, A. L., and Ra n d a ll, R. Z.: Protein measurement with the Folin phenol reagent. J. B iol. Chem. 293: , Lu b r a n, M. M.: The measurement o f total serum proteins by the Biuret method. Ann. Clin. Lab. Sci. 8: , NEURATH, A. R.: Interference of sodium ethylenediaminetetraacetate in the determination of proteins and its elim ination. Experientia 22:290, Parvin, R., Ra n d e, S. V., and Ve n Kitasu bra- MANIAN, T. A.: On the colormetric Biuret m ethod of protein determ ination. Anal. Biochem. 12: , Pea r so n, O. H., H a stin g s, A. B., and Bu n tin g, H.: Metabolismofcardiacmuscle:utilization of C14 labelled pyruvate and acetate by rat heart slices. Amer. J. Physiol. 158: , 1949.
5 PR O TEIN ANALYSIS O F SERUM AND H EA RT Pe ter s, T.: Proposals for standardization of total protein assays. Clin. Chem. 24: , Va n H o l d e, K. E.: Physical Biochemistry. Prentice-Hall, Englewood, NJ, W a d d ell, W. J.: A simple ultraviolet spectrophotometric method for the determination of protein. J. Lab. Clin. Med. 45: , Warburg, O. and Ch r istia n, W.: Biochem. Zeitschr. 320: , W ATSON, D.: Albumin and total globulin fractions o f blood. Adv. Clin. Chem. 5: , W EICHSELBAUM, T. E.: An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Amer. J. Clin. Path. 26:40-49, Z ishka, M. J. and N ishim ura, J. S.: Effects of glycerol on Lowry and Biuret methods of protein determination. Anal. Biochem. 34: , 1970.
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