Detection and Concentration Measurements of Proteins Adsorbed

Chapter 21

Downloaded by PENNSYLVANIA STATE UNIV on August 6, 2012 | http://pubs.acs.org Publication Date: June 3, 1992 | doi: 10.1021/bk-1992-0492.ch021

Detection and Concentration Measurements of Proteins Adsorbed onto Polystyrene and Poly(styrene—acrolein) Latexes Stanislaw Slomkowski and Teresa Basinska Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

Described is modification of the standard Lowry method, designed for determination of protein concentration in solution, in a manner enabling determination of protein adsorbed onto latex particles. Applicability of this method has been checked for human serum albumin (HSA) and gamma globulin (IgG), and for rabbit gamma globulins (IgGF and IgGFgD, antibodies against human fibrinogen and fragment D of human fibrinogen, respectively) adsor bed onto polystyrene, poly(styrene/acrolein), and poly(styrene/acrolein) • rhodamine 6G latex particles. The development of a simple and r e l i a b l e method, enabling detection and measurement of the concentration of proteins immobilized on the surface of latex p a r t i c l e s , i s important for applications of latexes in the l i f e sciences. Some examples were described recently i n monographs edited by Guiot and Couvreur (1) and by Rembaum and Tokes (2 ). Papers collected i n the mentioned monographs, concentrated mainly on new drug delivery systems, diagnostics, and c e l l sorting, represent only a small f r a c t i o n of a l l papers published within the l a s t decade on applications of latexes i n medical, veterinary, and plant disease diagnostics and treatment. Immobilization of b i o l o g i c a l l y active compounds on latex p a r t i c l e s i s useful for v i s u a l i z a t i o n of reactions involving proteins (e.g. i n the simple agglutination t e s t s (3-8)) and i s helpful for i s o l a t i o n of reaction products, selected macromolecules and c e l l s (9-19)Latexes are used also as c a r r i e r s transporting drugs (often proteins) to organs and as devices regulating the drug release (20-26). In b i o l o g i c a l studies and medical applications, latex p a r t i c l e s usually f u l f i l l the a u x i l i a r y functions; the main functions are related to the immobilized proteins. Thus, i t i s essential to have a simple method enabling the easy and fast detection of immobilized proteins and determination of t h e i r concentration.

0097-6156/92/0492-0328$06.00/0 © 1992 American Chemical Society In Polymer Latexes; Daniels, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

21. SLOMKOWSKI AND BASINSKA

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Methods Used f o r Determination of the Concentrations of Immobilized Proteins Unfortunately, almost a l l methods described i n the l i t e r a t u r e were developed for the detection and quantification of proteins i n solution (27-28). The few, which could be used also f o r immobilized proteins (e.g. radiochemical or Kjeldahl methods) have rather limited applicability. Radiochemical method can be used only for proteins l a b e l l e d with radioisotopes (cf examples given i n Ref. 29-34), require special equipment, and access to c e r t i f i e d laboratories. The Kjeldahl method, which consists of determination of nitrogen (constituting about 16 wt% of proteins), i s not very sensitive (usually more than 5 mg of protein i s required i n the analyzed sample (35)) and can not be used for latexes made from polymers containing nitrogen. Today, the most common approach i s based on determination of the difference between the protein content i n the i n i t i a l solution, used for immobilization, and i n the supernatant, obtained after removal of latex p a r t i c l e s with attached proteins (36-42). The protein content i n solution ( i n i t i a l solution or supernatant) could be determined by UV spectroscopy (absorption at 280 nm due to the tryptophan) or by other well known methods (e.g. ninhydrin (27), Lowry (43), or Bradford (44) method). However, determination "by difference" i s accompanied with an error that i s a combination of the errors of each separate measurement. In cases when immobilization i s carried on so that only a small f r a c t i o n of the i n i t i a l protein i s immobilized, the quantity of adsorbed protein may be within the r e s u l t i n g error. Moreover, one can not always be sure that the whole "missing" protein has been attached to latex, especially when supernatants are additionally p u r i f i e d from the traces of latex p a r t i c l e s by f i l t r a t ion. The d i f f i c u l t i e s connected with the determination of immobilized proteins "by difference" inspired us to investigate the p o s s i b i l i t y of adapting the methods designed primarily for measurements i n solut i o n to the d i r e c t determination of proteins adsorbed onto latex p a r t i c l e s . A l l methods based on dye binding to protein (e.g. the Bradford method) were excluded because measurement of the concent r a t i o n of dyes, immobilized as complexes with proteins on latex p a r t i c l e s , would pose no fewer problems than the d i r e c t determination of adsorbed proteins. The most promising methods were based on reactions of proteins with soluble, low molecular weight compounds yielding soluble products strongly absorbing i n the v i s i b l e region. Several years ago, Eskamani et a l used the ninhydrin method for the determination of proteins immobilized on collagen (45). We wanted t o check whether the c l a s s i c a l Lowry method (43), s t i l l very often used f o r determination of proteins i n solution and about 100 times more sensitive than the ninhydrin method, could be used also for determination of immobilized proteins. The Lowry method i s based on the reaction of protein (P) with Cu^ cations under basic conditions. At these conditions proteins are present i n polyanionic form. To the protein-(Cu2+) complexes a mixture of phosphomolybdic and phosphotungstic acids i s added. +

n

In Polymer Latexes; Daniels, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

330

POLYMER LATEXES

The reactions are schematically i l l u s t r a t e d by Equations (1) and (2). 2

P -

2 +

2

3 P~

C

+

• Cu

2 +

+

P-

12Mo0

3

3 P

. cu

2

^

u

+

• H P0 3

+

4

Cu (P0 ) 3

2 +

4

2

(1) 12W0 . H P0 3

+

3

6Mo 0 2

5

4

+

6W 0 2

5

(2)

2-


where P

denotes the negatively charged protein segment.

The tungstic and molybdic pentoxides eventually formed in t h i s process strongly absorb i n the v i s i b l e region (maxima at 750 nm). The protein determination by the Lowry method i s i n d i r e c t , depends on the nature of protein, and usually need c a l i b r a t i o n with standard protein solutions. The f i r s t attempt to use the Lowry method for determination of immobilized proteins was reported by Bunting and L a i d l e r (46) for acrylamide supports. However, the r e s u l t s were negative, presumably because of interference by free acrylamide apparently s t i l l present in the gel. We wanted to investigate whether the Lowry method could be used for the direct determination of proteins adsorbed on the surface of polystyrene latex p a r t i c l e s , the common polymeric supports for proteins i n diagnostic t e s t s , and on the surface of poly(styrene/acrolein) latex p a r t i c l e s . It i s known that latexes with aldehyde surface groups are convenient c a r r i e r s because they can immobilize proteins without p r i o r a c t i v a t i o n (12-14,47,48)For immobilization we have chosen proteins which are commonly used i n diagnostic t e s t s , namely gamma globulins (human and rabbit), and proteins often used in protein - polymer studies, namely human serum albumins. Preliminary r e s u l t s of the determination of gamma globulins and albumin at the surface of the polystyrene latexes are reported i n a paper (Basinska,T., Slomkowski.S. J.Biomater.Sci.Polym.Ed. i n press). In t h i s paper we w i l l concentrate on the comparison between detection of proteins immobilized on poly(styrene/acrolein) latex p a r t i cles, capable of binding proteins covalently and detection of proteins adsorbed onto polystyrene latex p a r t i c l e s , for which attachment i s only due to hydrophobic interactions. We w i l l also present some preliminary r e s u l t s on the determination of proteins adsorbed on the surface of a latex containing rhodamine G6, a dye used i n the synthesis of colored and fluorescent latexes e s p e c i a l l y s u i table for b i o l o g i c a l studies.

Synthesis of Latexes and t h e i r Characterization The polystyrene latexes were synthesized by emulsifier-free polymerization following the procedure proposed by Okubo et a l (49). The detailed description of the synthesis and p u r i f i c a t i o n of latexes i s given i n our previous paper (Basinska,T., Slomkowski,S. J .Bvomater.Sci.Polym.Ed. i n press^. The average size and size d i s t r i b u t ion of the latex p a r t i c l e s (D /D , where D and D denote the weight and number average diameters) were determined by scanning electron microscopy (JEOL 35C 200 A apparatus). The concentration of a c i d i c groups on the surface (A~) was determined by conductometric w

n

w

n

In Polymer Latexes; Daniels, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

21. SLOMKOWSKI AND BASINSKA

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331

t i t r a t i o n (cf e.g. Ref. 50). The composition of the polymerization mixture and values of the parameters characterizing the latex part i c l e s are summarized i n Table I.

Table I. Latexes Used f o r Protein Adsorption

Reaction


mixture

Weight

8

9

MI Styrene

9.06

Mil Styrene Acrolein

9.06 0.84

Mill Styrene Acrolein Rhodamine 6G

9.06 0.50 0.017

3

D n pm

D /D w n

0.52

1.004

3.38

-

0.36

1.002

2.57

1.76

0.30

1.30

[A"].10 2 mol/m

6

[-CH0]-10

6

2 mol/m

0.044 g ^ ^ O g and 102 g of H2O was used in each synthesis.

The poly(styrene/acrolein) latexes were obtained by emulsifier-free r a d i c a l emulsion-precipitation polymerization. The i n i t i a l concentrations of monomers and i n i t i a t o r (l

Detection and Concentration Measurements of Proteins Adsorbed

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