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Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A.

Ito L, Kobayashi T, Shiraki K, Yamaguchi H - J Synchrotron Radiat (2008)

Bottom Line: The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined.The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation.The addition of such additives appears to promote the crystallization of proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan. len@ksc.kwansei.ac.jp

ABSTRACT
Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

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Concentration range of precipitant in the reservoir solution in the presence of additives in which hemoglobin crystals were formed. Hanging droplets were made by mixing 1 µl of 40 mg ml−1 hemoglobin solution with 1 µl of reservoir solution. The reservoir solutions contain 5.0–40.0% PEG 3350 at 2.5% intervals, 0.2 M additives and 0.1 M sodium phosphate (pH 6.5). The bars show the precipitant concentration range in which crystals were formed.
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fig1: Concentration range of precipitant in the reservoir solution in the presence of additives in which hemoglobin crystals were formed. Hanging droplets were made by mixing 1 µl of 40 mg ml−1 hemoglobin solution with 1 µl of reservoir solution. The reservoir solutions contain 5.0–40.0% PEG 3350 at 2.5% intervals, 0.2 M additives and 0.1 M sodium phosphate (pH 6.5). The bars show the precipitant concentration range in which crystals were formed.

Mentions: Fig. 1 ▶ shows the results of crystallization of hemoglobin in the presence of additives with an initial concentration of 0.1 M in the hanging droplet at pH 6.5. The control experiment showed that hemoglobin crystals were obtained in the range 15.0–35.0%(w/v) of PEG 3350 in the absence of additives. In contrast, the concentration conditions of precipitant expanded in many cases in the presence of the additives. The use of Glu, Arg and glycine amide (GlyAmd) as additives was most effective for the expansion of the conditions of the precipitant concentration. When the concentration exceeded the precipitant by more than 37.5%, the hemoglobin solution aggregated in the absence of any additives, whereas single crystals appeared in the presence of Lys, Arg and GlyAmd, even at 40% of PEG 3350. In the presence of Gly, Glu and Lys–Glu, the concentration range in which crystals were obtained was expanded to a lower concentration. All the crystals had similar morphologies. In addition, the crystals formed more rapidly in the presence of the additives.


Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A.

Ito L, Kobayashi T, Shiraki K, Yamaguchi H - J Synchrotron Radiat (2008)

Concentration range of precipitant in the reservoir solution in the presence of additives in which hemoglobin crystals were formed. Hanging droplets were made by mixing 1 µl of 40 mg ml−1 hemoglobin solution with 1 µl of reservoir solution. The reservoir solutions contain 5.0–40.0% PEG 3350 at 2.5% intervals, 0.2 M additives and 0.1 M sodium phosphate (pH 6.5). The bars show the precipitant concentration range in which crystals were formed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2394812&req=5

fig1: Concentration range of precipitant in the reservoir solution in the presence of additives in which hemoglobin crystals were formed. Hanging droplets were made by mixing 1 µl of 40 mg ml−1 hemoglobin solution with 1 µl of reservoir solution. The reservoir solutions contain 5.0–40.0% PEG 3350 at 2.5% intervals, 0.2 M additives and 0.1 M sodium phosphate (pH 6.5). The bars show the precipitant concentration range in which crystals were formed.
Mentions: Fig. 1 ▶ shows the results of crystallization of hemoglobin in the presence of additives with an initial concentration of 0.1 M in the hanging droplet at pH 6.5. The control experiment showed that hemoglobin crystals were obtained in the range 15.0–35.0%(w/v) of PEG 3350 in the absence of additives. In contrast, the concentration conditions of precipitant expanded in many cases in the presence of the additives. The use of Glu, Arg and glycine amide (GlyAmd) as additives was most effective for the expansion of the conditions of the precipitant concentration. When the concentration exceeded the precipitant by more than 37.5%, the hemoglobin solution aggregated in the absence of any additives, whereas single crystals appeared in the presence of Lys, Arg and GlyAmd, even at 40% of PEG 3350. In the presence of Gly, Glu and Lys–Glu, the concentration range in which crystals were obtained was expanded to a lower concentration. All the crystals had similar morphologies. In addition, the crystals formed more rapidly in the presence of the additives.

Bottom Line: The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined.The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation.The addition of such additives appears to promote the crystallization of proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan. len@ksc.kwansei.ac.jp

ABSTRACT
Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

Show MeSH
Related in: MedlinePlus