Effect of TRIS buffer on the biuret reaction

SHORT

Effect

COMhlUNICA'I'IC)NS

of TRIS

buffer

453 on the

biuret

reaction

The biuret reaction was found to be satisfactory for cletermining protein concentration except for those solutions in which tris(liyclrox_ymctl~~l)amino~nctl~anc (TR~s) was prcscnt as the buffer. In these solutions, the sensitivity of the method was reduced and the results were gcncrally poor. Otllers have used the biurct method to cletcrmine pr0tein in solutions buffered with 7x1s Ien, but they did not indicate if *rr
Alkaline copper(I1) tartrate solutions were prepared as clcscribecl by Wmcrrs~xRAUM~. TRIS (General Biochemical and Sigma) was shown by its infrarecl .spectrum to be free of amide linkages. Electrophoretically pure bovine plasma and serum albumins (BSA) (Sigma and Calbiochem) were usecl interchangeably throughout the experiments. Titrations were carried out by the method of continuous variations. Equal volumes of alkaline copper(I1) tartrate and protein solutions were usecl and the resulting solutions were read with a Beckman DU spcctropliotometer in I-cm cells at 555 nm. Copper(I1) reagent concentrations of 0.01 2 M, 0.024 M, and 0.04s M (I, 2, and 3 times the 0.3% CUSO.~~~HZO suggested by WHICHSELRAUM) were used for the titrations. Protein concentration was the variable in each titration. Results and disczcssio~t Since TRIS is a primary amine, it would be expected to react similarly to tllc ammonium ion. TRIS was founclto complex the coppcr(I1) to a much greater extent than the ammonium ion. The absorbance maximum for the -rRIs-copper(I1) complex was practically the same as for the protein-copper(I1) complex (see spectra in Fig. I). AWLS. CIri*?t. Ada, 44 (1969) 453-456

Wllcn a 0.00~) A4 col)per(II) solution was titrated with mIs or ammonium cllloriclc, with absorbance rcatlings at 555 11171, thcrc was essentially no colc.~rformation in the latter cast, wl~orcas there was obvious color formation with ‘TRIS, the absorlmnce maximum ocCurring ;it 21,2 : I nlolnr ratio of 'I'RIS to Coppcr(II). 'I'hcsc? data for 7x1s confirm earlier lindingslO.

0.0 500

450

550

600

650

X(v)

l:ig. (3)

I. Absorption spcxtra lllb’/l”l bovine scrun1

of o.oo,~ &f coppcr(I I’) with (I) 0.067 M NIGCI, (2) 0.067 IW albumin (led). Collcclitrations arc tl1osc of final solutions.

I .o

-

PROTIIN

Fig.

2. Titmtion

nnc1

ns~

ancl

ccntrations

05A

and

lria

--+x-,--

5!5A

and

NH,Cl

CONCINTRATION

of 0.006 0.0125

arc those

I 3.0

I

I

4.0

5.0

Img./ml.)

lW coppcr(l I) with NH.&Zl. Alxwrbancc of final solutions. &I

ant1

051

---Z-m-

I 5.0

1

1.0

TRIS,

bovim a ~clw111 :Ltbuniirl (13s~). 13s~ and 0.0125 fif at 555 nni was rcarl against EL wntcr blank.

TRIS,

Con-

comparison was made of the amount of interference with the biuret reaction proclucccl by identical concentrations of ammonium ion and of TRIS. The effect of the ammonium ion was negligible (Fig. a), but TRIS interfered, reducing the sensitivity of the protein determination umxl~eclly. The esperinient with 0.025 ik? TRIS shown in Fig. 3 was at about a 4: I TRIScopper(I1) ratio, which was double the amount of TRIS necessary to complex all the copper(I1) present. Nevertheless, the absorbance of the protein comples increased as a function of incressecl protein concentration, thus demonstrating that the TRIS comples was more labile than the protein comples. The sensitivity of the deterA

Aml. Chim. Ada,

++ (rg6g)

453-456

sxiowr

COhIitIUNICA’I‘IONS

455

mination decreased as the amount of -rrzIswas incrcascd over the range 0.005~0.025 M. T11e 7x1s concentration used by some workers was lower than the o.o25 Al mIs used most often in the present work and was probably not competing wit11 protein for nearly so I~HX~~coplxr(II)1-3.

1.0

0..

1..

0.0

1

’ 0.0

I

I

2.0

1.0 PlOTtIN

CONCtNlRATION

I

I

4.0

3.0

1

3.0

[mo./ml.)

Fig. 4. Titration of o.ooG M coppcr(IT). 0.01 2 iZCcoppcr(l strum albumin (x3.4) and 0.01 2 L%ITRIS. i\bsOrhLllCC at Conccntratiowi 2m2 tllosc of final solutions.

I). a~icl 0.02.) nr coppcr(li) with bovine Ilnl wns rC;Ld :rgainst a water blilllk.

55.5

To obtain satisfactory results under the conditions shown in Pig. 4, the copper(I1) concentration had to be twice the 0.012 M (0.3% CUSO.~*~I-IZC>)reagent The difference between the curves concentration suggested by \~EIcHSEI.RAU~I~. obtained for 0.024 M and 0.04s M copper(I1) reagent for up to 3 mg/ml final protein concentration was due only to the absorbance of the copper(I1) reagent. The concentration range over which the curve was linear was practically the same for 0.04s M copper(I1) as for 0.024 M copper(I1) in the reagent and was within the capacity of most spectrophotorneters. -4 Nd.

CllillL.

n

Ckl,

‘14

(rg6g)

45p+jG

SHOH’I

456

COI\f~~UNICATIONS

It was conduclecl that the conditions best suitecl for protein determination in tllc presence ot *mrs were (.I) :L molar ratio of TIzIs: coppcr(I1) of I : I or lower, and (2) ;t protein ran’&2 of o-6 mg/ml lxfore addition of the reagent [0.024 M copper(I ~
I-II?

StLlCly

WLS

SLll)~xJI-ted

in

pLrt

hy

U.S.

Public

Hcdtli

Service

Grant

081og.

I... I<. S-rE\YAI
T. I9. WiSIcIIsEt.l3hunl, Am. J. Cli~it. .l’dlaol., J. I,. I-EALL, J. A. SWSIIIZR, 1). G. BRANNON

(Rcceivccl :lrbd.

Septemlxr

C/ri,rL. :Iclcr,

14th,

IgGS)

.t.t (L(J65)) Lt5_p-.t56

HULL

Y’l’cclr. ~3rrZl., 7 (1gqG) .to. ANI> ‘.r. h3. .I.AIXrN, Ilto!‘g.

C/WJU.,

I (1962)

atO9.