Tài liệu Báo cáo khoa học: Prevention of thermal inactivation and aggregation of lysozyme by

Prevention of thermal inactivation and aggregation of lysozyme

by polyamines

Motonori Kudou1

, Kentaro Shiraki1

, Shinsuke Fujiwara2

, Tadayuki Imanaka3 and Masahiro Takagi1

1

School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan; 2

Department of Bioscience,

School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan; 3

Department of Synthetic Chemistry

and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

Proteins tend to form inactive aggregates at high tempera￾tures. We show that polyamines, which have a relatively

simple structure as oligoamids, effectively prevent thermal

inactivation and aggregation of hen egg lysozyme. In the

presence of additives, including arginine and guanidine

(100 mM), more than 30% of 0.2 mgÆmL)1 lysozyme in

sodium phosphate buffer (pH 6.5) formed insoluble aggre￾1gates by heat treatment (98
C for 30 min). However, in the

presence of 50 mM spermine or spermidine, no aggregates

were observed after the same heat treatment. The residual

activity of lysozyme after this heat treatment was very low

(< 5%), even in the presence of 100 mM arginine and

guanidine, while it was maintained at
50% in the presence

of 100 mM spermine and spermidine. These results imply

that polyamines are new candidates as molecular additives

for preventing the thermal aggregation and inactivation of

heat-labile proteins.

Keywords: protein misfolding; protein aggregation; poly￾amine; thermal inactivation.

Proteins fold into their unique native structure, even in vitro.

However, they tend to form undesirable and uncontrollable

aggregates during the unfolding and refolding processes,

both in the laboratory and even in their natural environ￾ment in living cells. Protein aggregation is a major problem

in the large-scale production of recombinant proteins [1–3],

as well as in living cells, where it may lead to the occurrence

2of fatal diseases [4,5]. Various techniques have been

developed to prevent the formation of protein aggregates.

One of the major approaches used to prevent protein

aggregation is the addition of small molecules to the

solution. This is a relatively simple method compared with

using chaperon systems [6–8].

The small molecular additives used to prevent the

formation of protein aggregates are classified as protein￾denaturing reagents or others. Denaturants, typically

guanidine and urea, weaken the hydrophobic intermole￾cular interaction of proteins [9,10]. Detergents, such as

Triton-X100 and SDS, are stronger protein-denaturing

reagents than denaturants [10,11]. Not only do these

reagents dissolve aggregates and inclusion bodies but they

also unfold the native structure of proteins. Accordingly, the

concentration at which this type of reagent is effective at

preventing the aggregation and inactivation of proteins is

3hard to determine.

Arginine (Arg) is a nondenaturing reagent that has been

used widely as an additive to prevent protein aggregation

[9–12]. Arg does not destabilize the native structure, having

only a minor effect on protein stability [11,13], and enhances

the solubility of aggregate-prone molecules. Because of its

beneficial properties, Arg has been used for various proteins

and situations. However, the effect of Arg and other

nondenaturing additives does not completely solve the

aggregation problem. The development of better additives

for preventing protein aggregation has been long awaited.

In this article, we focus on naturally occurring poly￾amines [putrescine, NH2(CH2)4NH2; spermidine, NH2

(CH2)3NH(CH2)4NH2; spermine, NH2(CH2)3NH(CH2)4

NH(CH2)3NH2] as small molecular additive candidates

for preventing heat-induced aggregation and inactivation of

4proteins. There are a large number of different polyamines

in hyperthermophiles [14–16], which suggests that poly￾amines have a biophysical role in the adaptation of

hyperthermophilic proteins to high temperature environ￾ments. Although it has been reported that polyamines bind

to biomolecules (DNA, RNA, and platelets) by electrostatic

interactions [17–19], at present no research has been

published regarding the role of polyamines on thermal

aggregation and inactivation of proteins.

Materials and methods

Materials

Hen egg white lysozyme and betaine/HCl were purchased

from Sigma Chemical Co. All amino acids, guanidine/HCl,

urea, putrescine/2HCl, spermidine/3HCl, and spermine/

4HCl were purchased from Wako Pure Chemical Industries

(Osaka, Japan). Micrococcus lysodeikticus for the kinetics

Correspondence to K. Shiraki, School of Materials Science,

Japan Advanced Institute of Science and Technology,

1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan.

Fax: + 81 761 51 1655, Tel.: + 81 761 51 1657,

E-mail: [email protected]

Abbreviations: DCp, heat capacity change; DH, enthalpy change;

DSC, differential scanning calorimetry; Tm, midpoint temperature

of thermal unfolding.

Enzymes: lysozyme (EC 3.2.1.17).

(Received 29 July 2003, revised 27 August 2003,

accepted 23 September 2003)

Eur. J. Biochem. 270, 4547–4554 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03850.x