ANALYTICAI

68, 358-370

HIOCHEMISTRY

Electrophoretic Sulfate

Behavior

Complexes

Amounts

This sulfate

report present

tein-dodecyl mational these spectra

describes

the

solution

structural changes and and circular dichroism of the

same

amounts

forming enhanced teins that

a separation.

of Various

Dodecyl

between and

the

the

Sulfate

amount

of sodium

electrophoretic

mobility

In order to determine the extent and to establish a correlation the electrophoretic spectra were of surfactants

it is apparent solution must

Optimum

separation and can be accurately

Dodecyl

in the Presence

relationship

in a sample

From the results obtained. fate present in the sample

of Protein

of Sodium

sulfate complexes. changes in the proteins

presence

( 1975)

behavior, for

as used that the he taken

experimental

a maximized determined.

obtained

range

of any between

pro-

conforany of

visible absorption proteins in the

in electrophoresis.

amount of sodium into consideration

conditions linear

heme

dodecyl of the

are of molecular

chosen

dodecyl when for weights

sulper-

attaining of pro-

A variety of proteins have been separated and their molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis. Since it is an extensively used technique, procedures have been employed to determine its reliability in terms of the charge of the native protein (l-3). the conformation of the native protein (3,4) and the range of protein molecular weights that can be accurately determined (5-9). Several of the experimental conditions that have been varied include acrylamide concentration (5,6.8). amount of cross-linker (5,6), addition of urea to the system (6,8,lO), amount of a reducing agent such as /X-mercaptoethanol (I I), amount of N, N, N’, N’-tetramethylethylenediamine (12) and the use of disk electrophoresis (9). Little information has been reported on the effect of the amount of SDS present ( 13). Shapiro et nl. first observed that the relative mobilities (RM) of various proteins are dependent on their molecular weights in the presence of SDS ( 13). This dependency on molecular weight is based on the ’ Address

correspondence

to this

author-

at Ohio

358 Copyright All rights

i-1 lY75 by Academic Press, Inc. of reproduction in any form reserved.

University

ELECTROPHORETIC

MOBILITY

OF

PROTEIN-SDS

359

fact that DS binds to protein5 aon a gram to gram ratio ( 14.15). Therefore the charge to mass ratio ((>/,?I) of these proteins is the same at a specific binding ratio, and thus their migration in a polyacrylamide-gel medium under the influence of an electric field depends on the length of the protein complex chain which is a function of the molecular weight of the protein ( 16). Assuming there are different binding ratios, it would seem that the RM would be affected due to the influence of the binding ratio on (J/W. Besides the effect on c~/nz, SDS can also alter the structure of the protein. which is another important factor that controls the mobility of a protein-DS complex. Therefore the extent to which various amounts of SDS alter the structure must be considered. Information concerning any conformational changes can be obtained by observing the spectral properties of protein-DS complexes with absorption spectrophotometry and circular dichroism. Studies have been done concerning the effect on the visible absorption of heme proteins in certain denaturants such a:, SDS. urea and organic solvents ( 17-30). These spectral changes are attributed to the denaturation process. Circular dichroism has been trsed to determine the effect of these denaturing agents on the secondary structure ot proteins (2%76). The purpose of this investigation was to obtain various binding ratios of SDS to protein under constant experimental conditions and determine the effects of these ratios on the /