Marine Chemistry, 19 (1986) 1--7

Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands

A COMPARISON OF PROTEIN ASSAYS FOR OYSTER LARVAL PROTEINS USING TWO DIFFERENT STANDARDS
FU-LIN E. CHU and BEVERLY B. CASEY

Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062 (U.S.A.)
(Received June 20, 1985; revision accepted December 11, 1985)

ABSTRACT Chu, F.-L.E. and Casey, B.B., 1986. A comparison of protein assays for oyster larval proteins using two different standards. Mar. Chem., 19: 1--7. Using bovine serum albumin and bovine gamma-globulin as protein standards, two methods, a dye-binding method and the Lowry protein assay were employed to estimate the protein contents in oyster larvae. It was found that when both methods were used to measure the same concentration of the same larval protein sample, the Lowry method persistently generated higher calculated protein value than the dye binding method. The effect of trichloroacetic acid (TCA) and strong alkali (1 N NaOH) on these two protein assays was also determined. We found that TCA cause some interference with the dyebinding method.

INTRODUCTION

The Lowry method (Lowry et al., 1951) of protein assay is widely used, not only in the medical field, but also in the area of marine science (Giese, 1967; Scott, 1980; Ransch, 1981). Recently another protein determination method which involves the binding of Coomassive Brilliant Blue G-250 to protein, originally described by Bradford (1976), has been reported to be suitable for protein estimation in clinical assays (Bio-Rad Laboratories, 1979), for crude adrenal gland extracts (Pollard et al., 1978), and for particulate protein measurement in oceanographic samples (Setchell, 1981). This assay is recommended as a potential alternative for the Lowry method because of its simplicity and less interference from most common reagents (Bradford, 1976; Bio-Rad Laboratories, 1979; Setchell, 1981). Both Setchell (1981) and Pollard et al. (1978) reported obtaining equivalent results from samples measured by Coomassive Blue (dye-binding) and Lowry methods. We considered using this assay as a replacement for the Lowry method to determine the protein content in oyster larvae during development. However, the specific selectivity of the dye-binding method raises the question of whether this method is suitable for measuring the protein content of the crude extract from oyster larvae, since it has been reported that the sensitivity of the Coomassive dye to several purified proteins is not constant (Kley and Hale, 1977; Pierce and Suelter, 1977). There is also evidence indicating that the method of dye binding yields significantly lower values
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than the Lowry procedure in determining protein concentration of crude biological extracts (Chiappelli et al., 1979). For these reasons l~rior to applying the dye-binding method for our study, it was decided to compare both Lowry and dye-binding methods to measure the protein content ()i some larval protein samples. Protein larval samples were previously treated with 15 and 5% TCA for carbohydrate extraction. A strong alkaline reagent~ {1 N NaOH) was used to hydrolyze larval protein. Therefore, the effect of TCA and NaOH on the reagents of both Lowry and dye-binding methods was also assessed. METHODS AND MATERIALS

Preparation of oyster larual protein extract

Freeze-dried oyster larvae (0.1--0.4 g) were homogenized with 10--15 mJ of distilled water prior to carbohydrate and protein extraction. The oyster larval tissue homogenate was first treated with 15 and 5% trichloroacetic acid (TCA), respectively, to extract the total carbohydrate (Holland and Hannant, 1973). The mixture was centrifuged and the supernatant saved for total carbohydrate analysis. The residue (bound protein)was then hydrolyzed with 1--2 ml of 1 N NaOH at 100 -+ IC for 2 h. The hydrolyzed mixture wa~ centrifuged and the superna~ant used for the protein determination. REAGENT PREPARATION

Protein standards
Protein standards, bovine gamma-globulin {BGG) and bovine serum albumin (BSA), from Bio-Rad Laboratories (U.S.A.) were used.

Dye-binding reagent
For simplicity and consistency, a commercially prepared dye reagent from Bio-Rad Laboratories was used. The dye reagent, Coomassive Brilliant Blue G-250, was prepared to the concentration recommended by Bio-Rad Laboratories (1979), Bradford t 1976) and Setchell (1981}.

Lowry protein reagent

All of the reagents for L o w r y protein assay were prepared as described by L o w r y et al. (1951) and Hartree (1972). PROTEIN ASSAY For the dye-binding assay, we followed the standard procedure described by Bio-Rad Laboratories. The proportion of dye reagent to protein sample

or standard was 5:0.1ml. The procedure described by L o w r y et al. (1951) was used for Lowry protein assay. Both bovine serum albumin (BSA) and gamma-globulin (BGG) were utilized as protein standards. Identical standard protein solutions were assayed by b o t h the dye-binding and L o w r y methods. The wavelengths used for measurement of the optical density in L o w r y and dye-binding assays are 650 and 595 nm, respectively. In order to determine the reproducibility of the results generated by these two methods, two different concentrations (volumes) from the same larval protein sample were measured by the same procedure for protein content. Larval protein samples (25--100#1) were used for protein assay. Sample volume less than 100 #1 was adjusted to 100 pl with 1 N NaOH which was the reagent used for hydrolyzation of larval protein. The means of triplicate measurements are reported. For the determination of the effect of TCA and NaOH on the t w o protein assays, different concentrations of TCA (5--15%) and NaOH (0.05--1N) were separately added to proteins of similar concentration. Human serum albumin and bovine gamma-globulin were used as protein samples. Optical density of the protein samples was determined after mixing with Lowry or dye-binding reagents.
RESULTS AND DISCUSSION

The BSA and BGG standard curves generated by dye-binding and L o w r y m e t h o d s are shown on Fig. 1. The response curves to BSA and BGG for Lowry protein assay are very similar, in agreement with the finding of Pollard et al. (1978). For the dye-binding technique, the response to BSA is
~2T

,0q

/
40 PROTEIN 80 CONCENTRATION i 120 (/4g) 160

O2

0 0

Fig. 1. Response of proteins in the Lowry and dye-binding protein assays: (o), bovine serum albumin, ([]), bovine gamma-globulin. The curves represent the mean value of triplicate samples.

greater than to BGG as is also reported in the c o m p a n y literature (Bio-Rad Laboratories, 1979). It has been reported that the sensitivity of the dyebinding m e t h o d to various proteins varied greatly (Kley and Hale, 1977; Pierce and Suelter, 1977). This variability was suggested to be caused by different binding affinities of dye to different proteins. The protein contents of ten oyster larval samples as estimated by the dye-binding and Lowry methods are summarized in Table I. The dye-binding technique yielded consistently lower protein concentrations for all ten oyster larval protein samples. This could be seen from the data generated from the larval protein samples measured by these two methods. In measurements of identical concentrations of the same larval protein sample, the Lowry m e t h o d persistently produced a higher calculated protein value than the dye-binding method. When each method was used to measure two different concentrations of the same larval protein sample, the results obtained from the L o w r y protein assay were less variable than those from the dye-binding method. The variation of the results between dilutions of the same larval protein samples ranged from 3 to 17% in the dye~binding m e t h o d and 0 to 14% in the Lowry method. Our results agree with the findings of Chiappelli et al. (1979) that the protein concentration of crude cell and tissue extracts measured by the dye-binding technique produce significantly lower values than the Lowry procedure. However, the ratio of OD values or protein concentrations obtained from these two methods is not constant (2.16 + 0.74) (Table I). Constancy of the ratio of protein values was reported b y Chiappelli et al. (1977), when the two methods were used to estimate the protein content in human tissue and subcellular extract~ Our finding contradicted the results obtained by Setchell (1981) and Pollard et al. (1978), who reported equivalent values when dye-binding and Lowry protein assays were used to estimate total protein concentration. The reason for the lower protein values produced in the dye-binding m e t h o d is not certain, however, it does not appear to be due to insensitivity to a class of small peptides (Chiappelh et al., 1979). Although the dye-binding technique is simpler and more rapid than the L o w r y procedure, the selective binding of dye to various proteins and the production of low values for total protein measurement, as f o u n d in this and other studies (Kley and Hale, 1977; Pierce and Suelter, 1977)~ may make this technique inappropriate for total protein determinations. The results obtained from the determination of the effect of TCA ann NaOH on the two protein assays is presented in Table II, where means + standard deviations of triplicate samples are given. Sodium hydroxide (NaOH) concentrations from 0.05 to 1 N did not affect the development of color in either method. TCA appeared to produce some change in OD when the dye-binding method was used to measure two different proteins (human serum proteins and bovine gamma-globulin). The effect of TCA on these two proteins is approximately of the same order of magnitude.

TABLE I

C o m p a r i s o n o f t h e p r o t e i n c o n c e n t r a t i o n s o f larval p r o t e i n s a m p l e s as e s t i m a t e d b y t h e d y e - b i n d i n g a n d L o w r y m e t h o d s Dye-binding BSA equivalent (ug) 0.0 0.0 0.0 13 0.0 0.0 0.0 7.5 0.0 0.0 0.0 3 0.0 0.0 7.5 25.0 0.0 0.0 0.0 0.0 0.0 6 6 16 29 53 0.25 0.43 0.11 0.21 0.13 0.26 4 14 0.15 0.26 10 22 0.19 0.33 56.0 94.5 45.0 76.0 73.5 123.0 34.0 61.5 39.0 76.0 10 18 0.18 0.34 53.0 97.5 14 27 0.10 0.20 31.5 60.0 6 18 0.15 0.26 45 76 40.5 70.0 28.0 54.0 49.0 91.0 52.0 91.0 40.5 72.0 67.5 117.0 29.2 57.0 35.0 72.0 18 36 0.25 0.44 73.5 125.0 67.5 117.0 0.0 14 0.17 0.305 51 88 46.5 82.0 3.4 2.7 1.9 2.0 2.1 2.0 0.91 1.18 2.0 2.6 2.1 2.2 2.5 2.3 1.39 1.40 1.57 1.75 3.4 3.7
c5~

Larval protein sample BGG equivalent (~ug) BSA equivalent (ug) BGG equivalent (ug)

Sample volume used (pl)

OD at 590 nm

OD at 650 nm

Lowry

R a t i o o f OD value Lowry]dye binding

50 100

0.05 0.11

50 100

0.13 0.22

50 100

0.07 0.13

25 50

0.11 0.17

25 50

0.09 0.13

25 50

0.09 0.15

25 50

0.06 0.11

25 50

0.18 0.30

50 100

0.07 0.12

10

30 60

0.038 0.07

T A B L E II The e f f e c t o f TCA ( t r i c h l o r o a c e t i c acid) and NaOH o n the d y e - b i n d i n g and L o w r y p r o t e i n assays (values given are optical densities) Test ~/ Substances D y e binding OD 0.76 0.85 0.88 0.88 1.03 1.00 1.07 1.00 1.02 1.00 0.02 40.t)t : 0 : 0.01 /: 0.05 {~
t. 0.03 0

Test 2 a Lowry 0 D 0.93 0.94 0.94 0.97 i~01 ] ,03 0.03 -+ 0.04 -+ 0.01 0.03 +- 0.09 -+ 0.05 Dye binding OD 0.56 0,56 0.56 0.58 0.69 0.71
0.67 0.66

Lo~' ~y (3 t:) (J.5~ 0.64 0.56 0,55

),:'7

15% TCA 10% TCA 7.5% TCA 5.0% T C A 1 N NaOH 0.8 N NaOH 0.4NNaOH 0.2 N NaOH 0.05 N N a O H Control

~- 0.01 + 0.01 0.02 -+ 0 -+ 0,01 0.01

0.02 i 0.02

~ i: + i

0.ub 0.[~5 0.{)~ 0,0!

~-- 0 . 0 ~

{),59 ~ 0.(/5
0,5: t 0.0 (),57 ~ ~

-r 0.03
" '5

0.99 0.01 0.98 +- 0.02 0.99 -+ 0.01

i.O0 0.03

0.69 -+ 0.0l 0.67 0

q.54 ~ 0.0~, ~5:-:1 ~: C

aTest 1: 4 5 0 p g h u m a n s e r u m p r o t e i n w e r e a d d e d to each t e s t tube. T e s t 2 : 1 4 6 pg bovine g a m m a - g l o b u l i n w e r e a d d e d to each test tube.

CONCLUSIONS

(1) The dye-binding standard assay procedure as described in the Bio-Ra(l protein Assay Manual (Bio-Rad Laboratories, 1979) is not suitable for estimating total protein content for oyster larvae. (2) The reagents used in the Lowry method were relatively insensitive to TCA and NaOH, but TCA caused some interference to the dy~binding method.
ACKNOWLEDGMENTS

Contribution Number 1279 from the Virginia Institute of Marine Science. This work is a result of research sponsored in part by the NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant No NA81AA-D-00025 to the Virginia Sea Grant Program. The authors wish to thank Mr. Daniel Hepworth for technical assistance.

REFERENCES
Bio-Rad L a b o r a t o r i e s , 1979. B i o - R a d P r o t e i n Assay Manual. B r a d f o r d , M.M., 1976. A rapid and sensitive m e t h o d for t h e q u a n t i t a t i o n o f m i c r o g r a m q u a n t i t i e s o f p r o t e i n utilizing the principle o f p r o t e i n - d y e binding. A n a l Biochem.. 72: 2 4 8 - - 2 5 4 . Chiappelli, F., Vasil, A, and Haggerty, D.F., 1979. T h e p r o t e i n c o n c e n t r a t i o n o r crucie cell and tissue e x t r a c t s as e s t i m a t e d b y t h e m e t h o d o f dye binding: c o m p a r i s o n with t h e L o w r y m e t h o d . Anal, B i o c h e m . , 94: 160--165.

Giese, A.C., 1967. Some methods for study of the biochemical constitution of marine invertebrates. Oceanogr. Mar. Biol. Annu. Rev., 5: 159--186. Hartree, E.F., 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem., 48: 422--427. Holland, D.L. and Hannant, P.J., 1973. Addendum to a microanalytical scheme for the biochemical analysis of marine invertebrate larvae. J. Mar. Biol. Assoc. U.K., 53: 833--838. Kley, H.V. and Hale, S.M., 1977. Assay for protein in dye binding. Anal. Biochem., 81: 485--487. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folir phenol reagent. J. Biol. Chem., 193: 265--275. Pierce, J. and Suelter, C.H., 1977. An evaluation of the coomassive brilliant blue G-250 dye-binding method for quantitative protein determination. Anal. Biochem., 81: 478-480. Pollard, H.B., Menard, R., Brandt, M.A., Pazoles, C.J., Creutz, C.E. and Romu, A., 1978. Application of Bradford's protein assay to adrenal gland subcellular fractions. Anal. Biochem., 86: 761--763. Ransch, T., 1981. The estimation of micro-algal protein content and its meaning to the evaluation of algal biomass. I. Comparison of methods for extracting protein. Hydrobiologia, 78: 237--251. Setchell, F.W., 1981. Particulate protein measurement in oceanographic samples by dye binding. Mar. Chem., 10: 301--313. Scott, J.M., 1980. Effect of growth rate of the food alga on the growth/ingestion efficiency of a marine herbivore. J. Mar. Biol. Assoc. U.K., 60: 681--702.