A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies

William A. McGee, Lawrence J Parkhurst

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.

Original languageEnglish (US)
Pages (from-to)267-273
Number of pages7
JournalAnalytical Biochemistry
Volume189
Issue number2
DOIs
StatePublished - Jan 1 1990

Fingerprint

Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Hemoglobins
Light
Protein Unfolding
Oxyhemoglobins
Syringes
Heme
Denaturation
Cysteine
Tubing
Kinetics
Monitoring
Proteins
Flow velocity
Fiber optics
Fabrication

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies. / McGee, William A.; Parkhurst, Lawrence J.

In: Analytical Biochemistry, Vol. 189, No. 2, 01.01.1990, p. 267-273.

Research output: Contribution to journalArticle

@article{b577cb51f5e34cb183fcdf4ee03e3a7a,
title = "A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies",
abstract = "A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.",
author = "McGee, {William A.} and Parkhurst, {Lawrence J}",
year = "1990",
month = "1",
day = "1",
doi = "10.1016/0003-2697(90)90119-T",
language = "English (US)",
volume = "189",
pages = "267--273",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - A combined nuclear magnetic resonance and absorbance stopped-flow apparatus for biochemical studies

AU - McGee, William A.

AU - Parkhurst, Lawrence J

PY - 1990/1/1

Y1 - 1990/1/1

N2 - A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.

AB - A combined NMR and absorbance stopped-flow has been developed for monitoring the kinetics of biochemical reactions. We demonstrate its usefulness in following the alkaline denaturation of human hemoglobin. No glassblowing is required in the fabrication of the apparatus. Commercially available valves, syringes, tubing, and tubing connectors are employed whenever possible. Easily fabricated light guides are used to pipe light to and from the optical cell. The stopped-flow uses a 5-mm NMR tube followed by an optical cell with a 0.5-mm optical path length. This allows simultaneous measurements of NMR and absorbance changes. At a terminal flow velocity of 7.5 ml/s, the NMR and optical dead times were 60 and 260 ms, respectively. For the study reported here the oxyhemoglobin was labeled with a 19F probe attached to the β-93 cysteine. The native protein at pH 7 has an NMR spectrum consisting of a singlet, and the fully denatured hemoglobin sample at pH 12 has a spectrum consisting of three singlets. During the denaturation process another NMR peak appears rapidly and then decays away over the time course of the reaction. The absorbance changes at the high concentration employed for the NMR study (2.08 mm in heme) follow very nearly first-order kinetics. The events monitored by NMR, though in the same time frame as the optical changes, are of much greater complexity, and show the utility of multiple probes for monitoring protein unfolding.

UR - http://www.scopus.com/inward/record.url?scp=0025143321&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025143321&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(90)90119-T

DO - 10.1016/0003-2697(90)90119-T

M3 - Article

VL - 189

SP - 267

EP - 273

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -