Rapid preparation of native alpha and beta chains of human hemoglobin

Kay M. Parkhurst, Lawrence J Parkhurst

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

1. 1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is <0.1%. 4. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

Original languageEnglish (US)
Pages (from-to)993-998
Number of pages6
JournalInternational Journal of Biochemistry
Volume24
Issue number6
DOIs
StatePublished - Jun 1992

Fingerprint

Hemoglobins
Contamination
Mercaptoethanol
Photolysis
Carbon Monoxide
Assays
Ion exchange
Buffers
Gels
Oxygen
Oxidation
Kinetics
Lasers
Ion Exchange
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rapid preparation of native alpha and beta chains of human hemoglobin. / Parkhurst, Kay M.; Parkhurst, Lawrence J.

In: International Journal of Biochemistry, Vol. 24, No. 6, 06.1992, p. 993-998.

Research output: Contribution to journalArticle

@article{5e599d7174794a6c949d84bcd369157d,
title = "Rapid preparation of native alpha and beta chains of human hemoglobin",
abstract = "1. 1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5{\%} to less than 1{\%}, the limit of detectability. 3. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is <0.1{\%}. 4. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.",
author = "Parkhurst, {Kay M.} and Parkhurst, {Lawrence J}",
year = "1992",
month = "6",
doi = "10.1016/0020-711X(92)90109-E",
language = "English (US)",
volume = "24",
pages = "993--998",
journal = "International Journal of Biochemistry and Cell Biology",
issn = "1357-2725",
publisher = "Elsevier Limited",
number = "6",

}

TY - JOUR

T1 - Rapid preparation of native alpha and beta chains of human hemoglobin

AU - Parkhurst, Kay M.

AU - Parkhurst, Lawrence J

PY - 1992/6

Y1 - 1992/6

N2 - 1. 1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is <0.1%. 4. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

AB - 1. 1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is <0.1%. 4. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

UR - http://www.scopus.com/inward/record.url?scp=0026524308&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026524308&partnerID=8YFLogxK

U2 - 10.1016/0020-711X(92)90109-E

DO - 10.1016/0020-711X(92)90109-E

M3 - Article

C2 - 1612189

AN - SCOPUS:0026524308

VL - 24

SP - 993

EP - 998

JO - International Journal of Biochemistry and Cell Biology

JF - International Journal of Biochemistry and Cell Biology

SN - 1357-2725

IS - 6

ER -